introductory biography samples

What more do you need to know?

Ian doesn't take his bio too seriously but uses every character to highlight everything about him.

He includes his skills as a marketer and podcast host, who he is outside work as a dad, and what he can help you do. His smiles also give the bio a sense of humor and realness.

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The Best Short Professional Bios (Examples + Templates)

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Summary. To write a short bio you should first make an initial introduction introducing yourself in the first or first person. Your short bio should include your brand, your accomplishments, and your values and goals. Your short bio should be one to three short paragraphs or four to eight sentences long.

Knowing how to write a concise, informative, and interesting biography about yourself can help throughout various parts of the professional process. You can use your bio to capture the attention of potential employers or clients and convince them to choose to employ or work with you.

In this article, you’ll learn more about what goes into a short bio and how to write one, and you’ll also get to see some short bio templates and examples to help you get an idea of what yours should look like.

Key Takeaways

A short bio serves to introduce you, your achievements, and what you offer professionally to potential employers or clients.

It’s important to keep your bio brief so that readers stay engaged and will remember your main points.

You may need to adjust your bio for different audiences, as your clients may want to know different information than a recruiter would.

Talk about your skills and accomplishments in your bio, but don’t exaggerate them.

What Is a Short Bio?

How to write a short bio, what to include in a short professional bio, short bio examples, short bio templates, tips for writing a short bio, writing a short bio faq.

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A short bio serves as your introduction to the professional world. In terms of finding or expanding on your job, a bio will cover your:

Work history

Achievements

Any other relevant professional information

Think of it as a professional memoir that a hiring manager or consumer can read and understand quickly. It’s usually about one to three paragraphs depending on experience.

There’s an emphasis on being succinct when it comes to writing a professional bio. This is because a bio is supposed to be a preface to attract recruiter attention and incline them to reach out for more information. Many readers will get lost or bored with a lengthy bio.

Using a short bio can be helpful across very different industries, from marketing to accounting, from psychiatry to sales.

You’re probably familiar with providing short bios on social media websites and applications. While the information and skills you include in a professional bio may differ, the general formatting is similar.

There’s a lot of considerations to take into account when writing a short bio, and it can quickly become intimidating. Deciding what information is relevant and how to keep it near 140 characters is no small task.

If you’re having difficulty writing a short bio, follow the outline below to craft an introduction that engages your reader.

Make an initial introduction. You can’t jump right into everything you’ve done and what you want to do in the future before introducing yourself.

Your bio’s first sentence should begin with your full name in the third person or introduce yourself in the first person and continue to briefly outline your most notable skills and accomplishments. It’s a good place to state your current job and employer.

Go deeper with what motivates you. Once you’ve catchily illustrated who you are in your short bio, you can use the second sentence to describe your motivations for your work.

Stating what drives you to do the work you do is essential to employers and customers alike. Whether you work as a physician or fitness consultant , there’s a reason why this is your profession, and you should explain that in your short professional bio.

Describe your accomplishments. Your short bio is for detailing why you’re the ideal candidate to be trusted with handling an employer or consumer’s business. By describing your prior accomplishments, you let them know what you could offer as an employee and how you’ve succeeded in the past.

While you should avoid sounding braggy, the reader is looking for information about what your qualifications are , and your accomplishments generally measure these qualities.

Even though you could probably go on for ages about the details of your accomplishments, save that for an interview . In a short bio, only include the most impressive of your achievements to outline.

Accomplishments relevant to a short bio could include:

Impressive results on a project

Former promotions

Awards received in your field

Certifications received

Include contact information. The purpose of a short bio as either a business or a job seeker is to inspire the reader to reach out. Without contact information, this pursuit becomes futile. Make sure your short bio has some way to contact you at the end.

Relevant contact information may include:

Phone number

Professional networking profile

A short professional bio includes:

Your full name. You can choose to write your bio in the first person (I, me, my) or third person (he, she, they), but either way, you need to include your full name at some point. Branding doesn’t work so well without a brand name (i.e., you!)

Your brand. Of course, if you have an actual brand that you’re trying to market, you should include the brand name as well.

What you do. Summarize what you want the reader to know about what you do in one sentence — tricky, we know.

Your accomplishments. For a short bio, you can stick with just one major accomplishment from your professional life. Or, if you have a string of impressive achievements, try condensing all of them down to one sentence.

Your goals and values. Let the reader know what makes you tick — why do you do what you do and what do you hope to achieve with your work? People are compelled by a story more than anything else, so it’s important to get this part right.

Something personal (optional). If you have a quirky tidbit about yourself you’d like to include, go for it. Just make sure it doesn’t throw off te the tone of the rest of your bio.

Contact info (optional). If your bio is serving as a call-to-action to drum up business or get leads on job opportunities, it makes sense to include your contact information at the end of your bio. It’s not necessary if that information is available elsewhere on the page , though.

Entry-Level Job-Seeker Bio Example

Mitchell Morrison is an upcoming video producer and editor who believes in the art of visual organization. He is a recent graduate from the University of Washington and focused on post-production during his time studying there. He was introduced to the magical world of visual art production by watching his father work on editing commercials growing up and has been working towards his dream of becoming a video editor ever since. During his last year of college, Mitchell participated in a competitive internship with Digital Space Films. He was chosen out of 2,000 applicants based on his academic portfolio and personal statement essay. This internship was an incredible learning experience and resulted in three professional accreditations for music video editing. Mitchell currently lives in Seattle, Washington pursuing freelance opportunities and spending time with his Dog, Pikachu. To get into contact with Mitchell: MitchellMorrisonVideo.com/contact

Working Professional Website Bio Example

Lisa Kennedy is an experienced real estate professional. She knows how important a home is for long-term happiness and has invested her career in putting people in the house they’ve always dreamed of. Lisa was driven to pursue real estate from her passion for helping people during life-altering times, and a keen interest in high-end, luxury homes. She’s been working in the real estate industry for ten years and in that time has assisted over 3,500 people in finding homes. She was educated at the University of Los Angeles with a bachelor’s in business management. She’s worked for some of the most respectable Real Estate companies in Los Angeles and individually under her agency “Kennedy Homes.” Lisa has also been published in Real Estate Quarterly Magazine as the 2017 winner of the “Top Luxury Home Seller” award. Lisa loves the culture of Los Angeles and has been living there with her family of five since she graduated from college. She enjoys spending her free time exploring towns along the West Coast and swimming. If you’d like to get in touch with Lisa: Email: [email protected]

Professional Networking Profile Bio Example

Bianca Jones Marketing Manager Miami, FL The first step towards customer satisfaction is being reached by stellar product marketing, and that’s what I aim to provide. My professional experience as a product marketing manager has allowed me to assist many organizations in improving their sales margins and audience response to emerging products. I’ve brought dedication and positive results to the companies I’ve worked for because I am passionate about product perception, marketing, and business statistics. What drives a product to success interests and inspires me. I specialize in long-term growth strategies and audience outreach. In addition to eight years of experience in professional product marketing, I have also published two books on creating a career as a marketer called “What to Do After Your Bachelor’s” and “A Marketer’s How-To.” If you’re interested in learning more about how to market your business better, or just discuss more, feel free to contact me by email at [email protected].

Your first choice is whether you want your bio to be written in the third person or first person. These short bio templates show both options, and also include different ideas for what to include, and how. Feel free to pick and choose your favorite parts of each of the two.

[Full Name] is a [job title] who [believes/knows] in the power of [what you do]. [He/She/They] began their journey in [field] by [how you got started in the field], and now dreams of [what you hope to accomplish]. [His/Her/Their] biggest accomplishment to date has been [your biggest accomplishment]. [Full Name] lives in [where you live] and participates in [a hobby/interest]. To get in touch with [Full Name], call/email/message me on [how you’d like to be contacted].

I am a [job title] who helps [who you help] [what you help them do]. It’s my belief that [your unique perspective on the field]. In the past [# of years] years, I’ve [major accomplishment #1] through [how you accomplished it]. I have a passion for [your professional passion], but on the side, I also enjoy [personal passion]. Get in touch with me today at [contact info] — I look forward to talking with you about [what you want to talk to your readers about].

You have a firm grasp of the structure of a short bio and what to include. Now, you may need some tips for how to polish your short professional bio and make it stand out from the competition.

Be mindful of length. While you’re probably getting sick of hearing that your bio should be short, it’s good to keep in mind throughout the writing process. It’s easy to go off on a tangent while trying to include everything relevant or rationalize, making your bio too long.

Avoid this impulse. The point of a bio is that it’s limited. You want to intrigue the reader enough to inspire them to seek more information about you or your services.

Tailor your bio to your intended audience. Whether you’re using a short bio to attract a particular customer base or potential employer, tailoring it to fit their wants and needs is crucial. Consider your intended audience base and what they’re looking for in a candidate or service.

Be genuine. Your short bio should be an authentic representation of your traits, experience, and personality. People are repelled by what they interpret as stretching the truth. If you’re being received as disingenuous by the reader, they’ll probably move on.

Proofread. The only way to steer clear of errors in your short bio is by proofreading it. Imagine a hiring manager being completely interested in your bio.

They love what you have to say about yourself and find your prior experience enticing. That is, until they come across a mistake that clearly shows you didn’t do proofread or edit.

Include links to your portfolio, website, or networking profile. One way to circumvent the confining factor of keeping your bio short is by including links to more detailed sources.

This can be in the form of linking your portfolio or website to allow the reader to go deeper into your discussed skills if they please, without taking up more space in your bio.

Implement these links seamlessly into your bio by attaching them to anchor words that describe what clicking will lead them to.

Add some personality. You aren’t the only person who has an impressive list of accomplishments to put on a bio, so you’re going to need to find some additional ways to make an impression.

What should a short bio include?

A short bio should include your name, what you do, and your achievements. You should also include your company or product’s brand, if you have one, and your goals and motivations for doing what you do. This humanizes you and helps you stand out from the rest of the pack.

How long is a short bio?

A short bio is typically one to three paragraphs long. These should be short paragraphs though, as other experts say that between four and eight sentences is the ideal length for a short bio.

What makes a good bio?

A good bio is succinct and memorable. Readers don’t want to spend long reading about your professional and personal life, so go back and cut it down to the important parts multiple times after you draft it. You might be surprised at how little you actually need to include.

What should you avoid putting in a short bio?

You should avoid including anything negative or arrogate. It’s never a good idea to write anything negative about previous jobs or employers. Only include positive things in your professional short bio.

It’s important to include your achievements in a short bio, but there is a fine line between mentioning your achievements and bragging about them. Stick to the facts when talking about your accomplishments.

Fremont University – Building Your Professional Bio

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Sky Ariella is a professional freelance writer, originally from New York. She has been featured on websites and online magazines covering topics in career, travel, and lifestyle. She received her BA in psychology from Hunter College.

Don Pippin is an executive and HR leader for Fortune 50 and 500 companies and startups. In 2008, Don launched area|Talent with a focus on helping clients identify their brand. As a Certified Professional Resume Writer, Certified Digital Career Strategist, and Certified Personal Branding Strategist, Don guides clients through career transitions.

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How to Write a Short Professional Bio (With Examples and Templates)

3 key takeaways

• What a short professional bio is and why you need one
• What to include in a short bio to make it stand out and relevant to your audience
• Examples, templates, and a faster way to write your bio with Teal's Professional Bio Generator

In a world where just about everyone is represented online, your short professional bio often serves as a first introduction.

Often, it's what employers or other professionals will use to form their initial impression. And how you present yourself can help influence whether someone invites you to connect, interview you, or simply move on.

Below, you'll learn what you need to write your bio and how to actually write one. You'll also find templates and some short professional bio examples to use as a guide.

Looking for a quicker way to a polished bio? Sign up for Teal for free and try the Professional Bio Generator.

What is a short professional bio? 

A short professional bio is exactly what it sounds like—a short professional biography that introduces yourself and gives a brief overview of your career and accomplishments. It gives future employees, colleagues, and anyone else you're networking with a more well-rounded picture of who you are.  

While "short" can be subjective, length often depends on the platform and the audience you're sharing your bio with. There's no one-size-fits-all, and you'll tailor the length based on where it's being shared—whether that’s on your personal website, LinkedIn profile, Twitter bio, or your resume.

What’s the difference between a professional bio and short professional bio?

A professional bio and a short professional bio both summarize your career, qualifications, and achievements, but each caters to different contexts and needs. 

A professional bio is a detailed account—that can span several paragraphs—and covers your:

• Career background
• Most noteworthy achievements and impact or results
• Skills you've developed or are developing
• Some personal insights

This version is appropriate for professional websites, long-form pitch documents, or networking platforms (like in your LinkedIn About Me section) where a more thorough introduction is valuable.

On the other hand, a short professional bio is a concise version that filters the most important information from your professional bio into key takeaways. A short bio:

• Communicates your core qualifications
• Briefly touches on your professional persona
• Is ideal for your resume, author bio, or speaking engagements
• Can be used across social media profiles like Twitter and Instagram

What to include in a short bio 

Your short bio should summarize the key takeaways from a longer, more rounded-out professional bio. Think of it as kind of an elevator pitch that highlights what you'd want your target audience (who doesn't know much—if anything—about you) to understand about your career so far.

Here are some things you'll want to address.

• Level of education, if relevant to the audience
• Years of experience in your field, if relevant to the audience
• Your current or former career title
• Major professional accomplishments with impact or goals
• Professional experience, skills, and area(s) of expertise

Now, if you're a student or making a pivot, like transitioning into a new field where you might not have relevant work experience, you'll focus more on your educational achievements (think relevant awards, projects, or volunteer experience) and the skills and knowledge you're developing.

What to include in a short bio if you’re a student

Even if you're still in school, there's plenty of information you can include in your bio, like: 

• Where you're from
• What university, trade school, or boot camp you're attending
• Your area of study or intended major
• Any clubs, teams, volunteering, or other extracurriculars you're a part of 
• Any internships you've completed
• Your career goals 
• Your availability

Generate your professional bio with AI

Want to save time? You can also use the Teal to generate multiple versions of your short professional bio.

How to write a short bio

There's no right or wrong way to write your own bio. Think of this next section as a guide, and remember: short professional bios are unique to each person.

Here are the things you might want to consider if you're ready to write your professional bio:

1. Choose your voice

You have the option to write your short bio from either a first or third-person POV.

In the first person, you'll use  "I, me, and my." In the third person, you'll refer to yourself using your name and preferred pronouns.

As a general rule, using the third person is typically considered more formal, while using the first person is more casual and personal.

 First-person example : "I'm a bestselling author. My work has been published in The New York Times."

Third-person example: "Zane Smith is a bestselling author. His work has been published in The New York Times."

If you want to write and store multiple versions of your short professional bio in different tones or even with different information, use Teal to write and save as many versions as you need for free.

2. Choose your tone

Language can convey different moods. Depending on the context and your personal brand , your bio can sound more buttoned-up or more relaxed and conversational.

These two sentences describe the same person but showcase very different tones and writing styles: 

Example 1: “A recent graduate of the University of Pennsylvania, Sally is currently a consultant at XYZ Consulting based out of their New York office.” 

Example 2: “A recent UPenn grad, Sally swore off econ after years of hard classes only to end up as a consultant at a major firm — but don't worry, she loves it.” 

3. Start with your name and a current or recent role

This isn't a hard and fast rule, but to get your bio started, it's usually easiest to start with your name and current professional title or role. 

Example: “Zane Smith is currently the Demand Generation Manager at ABC Tech Company.”

If you're pivoting careers , you can list your most recent role and use your first sentence to say what you're looking to do next.

Example 1: “Previously the Demand Generation Manager at ABC Tech Company, Zane Smith is actively pursuing new opportunities in marketing management.” 

Example 2 : “Previously the Demand Generation Manager at ABC Tech Company, Zane Smith is now an aspiring 2nd grade teacher.” 

4. Share your accomplishments and achievements

Once you've introduced yourself and your current title, you can flow into your professional background, former job titles, and what you've done or aspire to do. Together, the first two sentences of your bio could read something like this: 

Example:  “Zane Smith is currently the Demand Generation Manager at ABC Tech Company. Previously, he was a Marketing Specialist at FGH Tech Company, where he grew paid traffic to their website by 500% year over year.” 

Pro Tip: Not sure where to start? Try the generative AI in the Teal Resume Builder . With Teal's AI, you can write individual achievements using a job description, keywords, or a custom prompt to articulate your accomplishments effectively and confidently.

5. Share your philosophy or “why” 

Self-promotion can feel incredibly uncomfortable, but it's important to make sure you don't undermine your value. Your “why,” the reason you do what you do every day, can be powerful and meaningful. Adding what gives you the spark to start each workday can set your own professional bio apart from others. 

Example: “Claire was inspired to pursue a career in elder law after volunteering in a nursing home throughout high school and college.” 

6. Add your personal touch

Even though your bio will be used in a professional capacity, it's okay to list a few personal details. We're more than just our jobs, and adding a few personal facts can help illustrate who you are outside of the office. 

Example: “When he's not working, Martin can be found tending to his spice garden and going on nature walks with his golden retriever.” 

Short professional bio examples

Examples of professional bios are invaluable because they provide a clear framework and inspiration for writing your own. They can also help you see how to condense years of experience into a few compelling sentences so you communicate the right details clearly, briefly, and full of impact. 

Short bio example for a personal website

"AUDREE KATE LOPEZ IS A FASHION STYLIST, EDITOR, INDUSTRY EXPERT AND INFLUENCER BASED IN NEW YORK. Audree began her professional career in the fashion departments at Glamour, O, the Oprah Magazine, Editorialist and Redbook magazines. She launched Audree Kate Studios in 2017 and worked on freelance projects at Marc Jacobs, Alice + Olivia, J.Crew and Club Monaco, joined the styling team at Alice + Olivia and became a contributing fashion editor for StyleCaster.com. Over the past few years Audree has styled for various publications, celebrities and retail brands. In 2016, Audree founded a digital course and community for fashion students called Fashion Fundamentals and has hosted workshops and classes around the country, and wrote an ebook titled Fashion Fundamentals."

Why it works: Audrey's bio immediately establishes her as a multifaceted professional within the fashion industry. It outlines her career trajectory and showcases broad experience and evolution, demonstrating growth and adaptability. The mention of initiatives and workshops adds a layer of approachability and commitment to education in her field, improving her appeal as an influencer and mentor.

Professional short bio example on LinkedIn

"Latina creator. Speaker. Tech Mentor. Christen is the Founder of the newly launched app Clara, a community that empowers creators through transparency, brand reviews, and discoverability. An industry vet, Christen has almost a decade of experience working with top content creators at social networking companies such as Instagram, TikTok, and Pinterest. She is a motivational speaker and mentor in the tech space who is passionate about helping people break into the industry through career advice and actionable content."

Why it works: This professional bio online is punchy and concise. It opens by noting Christen's titles and includes years of experience, giving her credibility while underscoring her heritage (which can resonate strongly with a diverse audience on LinkedIn). By focusing on her app, she not only showcases initiative but also aligns herself with trends in tech innovation. Rounding out her short bio are details of working with top content creators, establishing her as a seasoned industry pro.

Short bio example on social media

"Founder of Makelane, a private community for female DTC founders. 70K followers on TikTok where I analyze consumer brands"

Why it works: With only 160 characters to work with, Dulma’s Twitter bio is an example of brevity. It clearly outlines her two main pursuits: managing an online community and producing content for TikTok, making it easy for readers to gauge her interests and activities at a glance.

Company website short bio examples

"Meet our wonderful team We're a tight-knit group of curious creatures, always learning, and constantly seeking out new perspectives and ideas. Get to know our team—they're what makes Teachable, well, Teachable."

Why it works: "Meet our wonderful team" feels like a personal invitation to get to know the group, which can make visitors more inclined to engage. Describing them as "tight-knit" and "curious creatures" adds character and illustrates a vibrant and collaborative work environment. Highlighting that the team is "always learning" and "constantly seeking out new perspectives" positions Teachable as a dynamic and innovative company—committed to growth and evolution.

Tips for writing a short bio

Even though there aren't official rules for writing a bio, there are still some things you should strive for as well as steer clear of if you want to make the best possible impression. 

1. Tailor your professional bio to your audience

Whether it's potential employers, LinkedIn connections, Twitter followers, or visitors to your website, each platform and audience has its own expectations. Keep your information relevant and concise, focusing on what matters most to that intended audience.

2. Be authentic

Your bio is not the place to fudge the truth about who you are. Be honest about what you've done and where you're heading. Not telling the truth could bar you from achieving your goals and land you in hot water. 

3. Prioritize relevant information

We really hate to be the one to tell you this, but unfortunately, most people do not have the time or patience to read a five-paragraph essay about your life story. Sorry to break it to you! 

4. Be relatable

No matter how lofty your accomplishments are, staying grounded may help you establish stronger connections with others. This is where adding personal details and highlighting your personal brand can serve you well. Whether that's with a hobby, interest , or other role outside of work, being relatable can let others understand and get to know you better and determine if you would be a good fit for a company.  

5. Be professional

It's appropriate and expected for you to talk about your professional skills and accomplishments in your short bio. That said, there's a fine line between talking about your achievements and sounding arrogant. These two examples talk about the same accomplishment but come across differently: 

Example 1: “An accomplished pianist, Ryan was selected out of nearly 7,000 applicants to perform at the annual New York State School Music Association festival last spring.” 

Example 2: “An accomplished pianist, Ryan was the obvious choice to perform at the annual New York State School Music Association festival last spring (because he's the best).” 

Also, if you're revising your bio after a lay-off or termination, avoid sounding spiteful. As tempting as it might be, don't make negative statements or accusations in your short bio. Positivity and a good attitude will get you much further.

Short professional bio templates

If you prefer to plug and play or need a short bio in a pinch, don't worry; here are three short bio templates to get you started:

Short professional bio template for working professionals

[First name last name] is [currently/formerly] a [insert most recent job title] at [most recent company name] . A proud graduate of [school or university] , they were inspired to pursue a career in [field] after [explain what led to your decision to work in your industry] . Prior to working at [most recent job title], they were the [previous title] at [previous company], where they were responsible for [insert professional accomplishments here] . In their free time, they can be found [insert your favorite hobbies] . 

Short professional bio template for students

[First name last name] is a current [year] at [institution] majoring in [area(s) of study] . On campus, [First name] is actively involved in [activity name] , for which they [insert details about what you do for the activity you're involved in] . They most recently [interned/worked] at [organization name] , where they had the opportunity to [insert details about what you did on the job] . They intend to work in the [insert field name or industry] after graduation to follow their passion of [insert what you are passionate about] . In their spare time, they can be found [insert your favorite hobbies] . 

Short professional bio template for a gap in employment

[First name last name] is a [position/job title] with [number] years of experience in [specific skills or industry] . Following [briefly mention the reason for the employment gap] , [he/she/they] took some time off to focus on [briefly mention what they did during the gap, e.g., personal development, family obligations, health reasons] . During this time, [he/she/they] also [briefly mention any relevant skills or achievements acquired during the gap] . [Name] is now eager to return to the workforce and is excited to bring [specific skills/accomplishments] to [his/her/their] next role. With a proven track record of [list specific achievements or skills] , [Name] is confident in [his/her/their] ability to contribute to [specific industry/profession] and make an impact in [his/her/their] next position.

If you decide to use any of these templates, tweak it ever-so-slightly. Once you've finished entering your details, try adding a bit of personal flair.

Swap adjectives, remove anything that doesn't quite fit with your vibe or experiences, or append some more personal details as you see fit. Feel free to work off of any one of the bios from these resume examples to get a feel for how different styles can be used across roles.

Create your short professional bio with Teal

In a digital world where your online presence often speaks before you do, writing a good, short professional bio is more important than ever. It's the first glimpse potential employers, clients, or colleagues get of your professional life and persona, and the right presentation can open doors to new opportunities and connections. 

Teal's Professional Bio Generator saves time by speeding up the writing process with AI. Whether you're trying to impress potential employers, clients, or peers, Teal's generator adapts to your needs and experience, allowing you to create a unique short bio for any audience with one click.

Ready to write a professional bio with maximum impact in less time?

Frequently Asked Questions

What is an example of a short professional bio.

An example of a short professional bio is: "Katie is a seasoned marketing professional with over 10 years of experience in digital advertising strategies. She has helped numerous brands achieve their online marketing goals, leading to increased visibility and 3x revenue YoY."

How do you write an impressive short bio?

To write an impressive short bio, focus on your most significant achievements, relevant skills, and experiences that set you apart. Keep it concise and tailored to your audience to engage and leave a memorable impression. You can also use the Teal  AI Resume Builder  Generative AI to craft a bio that highlights your unique professional journey and skills in an engaging way.

What is a short biography about yourself?

A short biography about yourself should concisely summarize your professional background, major accomplishments, and skills. For example, "I'm an innovative software developer with 5 years of experience in creating scalable applications, known for my ability to troubleshoot complex issues and my commitment to delivering high-quality software solutions."

Emily Polner

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How to write a professional bio (with examples and templates)

• Rebecca Strehlow
• 11 min read

Get started by: Creating a website →  | Getting a domain →

Which three words would you use to describe yourself? Most of us have been asked this question, and many of us have fumbled through it awkwardly.

Coming up with a personal description can be daunting. But there are times when it’s essential - whether we’re updating our LinkedIn profiles, blogging for Medium or creating a business website of our own.

In this post, we’ll go over how to write a bio, step by step. To help guide you, we’ve also included a handy template, along with some professional bio examples for your inspiration. With these resources, you’ll find that writing a bio, as part of making a website , is much easier than you might think.

What is a bio?

Before you learn how to write a bio, you should have a clear understanding of what it is and why you need it.

In the world of literature, a personal biography can span the length of an entire novel, like Nelson Mandela’s Long Walk to Freedom or Malala Yousafzai’s I Am Malala . In the online world, however, a bio is a short paragraph in which you introduce yourself. Typically, people place bios on the About Us page of their professional website, as well as on their social media pages and other networking platforms.

What to include in a bio

Depending on your audience and goals, your bio can highlight your personal interests, your professional achievements or a mix of both. Here are some of the elements a bio might include:

Job title or workplace

University degree and other qualifications

Hometown or city of residence

Personal or professional goals

Mission statement and values

Skills and expertise

Interests and hobbies

The goal of writing a bio is to provide people with a snapshot of who you are. This is important for a variety of reasons, whether it’s drawing people toward your personal website or promoting your blog, attracting clients and business partners to your brand, or highlighting your achievements for job interviews.

How to write a short bio

The most effective online bios are both professional and concise. Here’s how to write a short professional bio that suits your website or brand:

Introduce yourself

State your company or brand name

Explain your professional role

Include professional achievements

Discuss your passions and values

Mention your personal interests

01. Introduce yourself

Begin your bio by stating your first and last name. If you’re writing in the third person, these should be the first two words of the paragraph. This makes your name easy for your audience to identify and remember. Your bio is a huge part of your personal branding efforts, and should start with a strong intro to make a positive impact.

02. State your company or brand name

Think about whether you want your bio to represent yourself on a personal level, or whether you’d like it to come across as more professional. If you have a personal brand or business - for example, a blog, freelance business or eCommerce site - be sure to mention your brand name at the beginning of your bio. Don’t be afraid if the name sounds simple or redundant. It’s perfectly fine, for instance, to say Mary Smith is the founder and CEO of Smith Digital.

Likewise, feel free to mention the name of another company or brand that you work for if you’d like to associate it with your professional accomplishments - e.g., Mary Smith is a consultant at Google and the founder and CEO of Smith Digital.

03. Explain your professional role

Next, briefly explain your current position. This is relevant whether you’re the founder of a company, a high-level specialist or a beginner in your field, and it can be similar to the description you have on your resume. Your website visitors won’t necessarily know what your job involves, so elaborating on your primary responsibilities helps paint a picture of who you are and what you have to offer. This can also be used, if needed for employment and recruitment opportunities. If you're a freelancer a strong bio can make all the difference in how successful sourcing work can be.

04. Include professional achievements

In addition to explaining what your job entails, highlight milestones that make you stand out. Even if you haven’t won an award or gained external recognition, you can discuss ways in which you’ve contributed to your professional role and touch on new ideas or approaches that you bring to the table.

05. Discuss your passions and values

Once you describe what you do and how you contribute to your role, you’ll need to explain the why . This is one of the most important elements to focus on as you consider how to write a bio.

Think about the values and passions underlying your work, as well as your professional philosophy. What gets you up in the morning? What’s the driving force behind what you do?

You can also think of this part of your professional bio as a kind of mission statement. Perhaps your mission is to serve others, contribute to society, grow your expertise or learn new skills. Whatever your reasons, expanding upon these ideas can help your audience get a better understanding of what truly matters to you. Don't be afraid to deploy storytelling in this part of creating your bio. Explore your narrative and then convey it.

06. Mention your personal interests

The most effective short bios will not only focus on your professional experience, but will also touch on what you like to do in your spare time. Consider mentioning:

Your family

Your hometown

Your hobbies

Side projects you’re working on

Transitioning to a more casual discussion of who you are outside of work is a great way to conclude your bio. This will present you as a more well-rounded person while making you relatable for your audience.

Professional bio template

As you go through the steps on how to write a bio, this handy template will help you get started:

Sentence 1: [Name] is a [job title] who [job description].

E.g., Lisa Green is an English teacher who teaches beginning to advanced literature courses for 10th and 11th grade students at Bloomfield High School.

Sentence 2: [Name] believes that [why you do the work you do].

E.g., Lisa believes that written and analytical skills are not only a fundamental part of academic excellence, but are also the building blocks of critical thinking in high school and beyond.

Sentence 3: [Name/pronoun] has [mention your achievements].

E.g., In addition to managing the English curriculum for the school, she runs an after school program where she works one-on-one with students.

Sentence 4: [Name/pronoun] is a [mention any relevant awards, training or honors].

E.g., She has also been nominated Teacher of the Year for two consecutive years.

Sentence 5: [Name/pronoun] holds a [insert degree] in [field of study] from [university].

E.g., Lisa holds a BA in Creative Writing and a Master’s Degree in Teaching from the University of Michigan.

Once you’ve filled in this template, put it all together into a single paragraph to create an initial framework for your professional biography. Note that you can shorten or expand upon this bio according to your unique needs.

Professional bio examples

Now that you know the basics of writing a professional bio, here are some short bio examples to inspire you. You can use these examples as additional templates for guidance as you craft your own personal biography.

Like the creators of these examples, you can place your bio on your personal or professional website and, later, revise the structure for other online platforms.

01. Bristol Guitar Making School

Of all the professional bio examples, Alex Bishop’s content exudes passion. Strategically placing the bio on the About page of his small business website , he highlights his skills and explains why he finds his work meaningful. In particular, we love his description of why he chose to pursue guitar making:

“​My passion as a guitar maker comes from a life-long obsession with making things. From a young age I have always tried to manipulate objects and materials in order to create something entirely different. I find that working with wood is a way for me to connect with nature. The simple act of shaping wood to make something functional or beautiful brings me endless satisfaction.”

He also lists his accomplishments and awards, adding credibility to his business and building trust among prospective clients.

02. Alexandra Zsigmond

As someone who has served as art director for both The New York Times' opinion section and The New Yorker , it's no surprise that Alexandra Zsigmond's bio is thorough and detailed. Providing statistics or reflections on the things she achieved in her career is a clever way to demonstrate her value without saying so directly. As she explains:

"She has collaborated with a roster of over 1000 artists worldwide and art directed over 4000 editorial illustrations. She is known for greatly expanding the range of visual contributors to the Times, drawing equally from the worlds of contemporary illustration, fine art, animation, and comics."

03. Amanda Shields Interiors

Amanda Shields provides us with another effective bio example on her interior design website. Importantly, she spices up her bio by explaining how home decor aligns with her personal life and why it’s so close to her heart as a mother and entrepreneur:

"After working as a product designer for numerous retailers over the years, and after I had my first child, I decided to take the plunge and start my own home staging business…. Coincidentally, a month later I discovered I was pregnant with my second child. I loved the new challenges I faced as a new entrepreneur and mom and it didn't take long for me to discover that this was my calling…. I felt the need to expand my business and launch Amanda Shields Interiors as its own entity to focus specifically on residential interiors and design."

By placing this content on her website’s About page, she provides potential clients with insights into her expertise and professional experience. She expands upon the choices she made along her career path, strategically making note of her achievements and acquired skills along the way.

Tips for writing a bio

As you write your bio using the professional template above, make sure to keep the following tips in mind:

Keep it concise: Your bio should be sufficiently explanatory, but it should also be short and to the point. A good rule of thumb is to keep each element of your bio - from your job description to your mission statement and hobbies - to about 1-2 sentences. That way, you’ll end up with a brief paragraph that holds your readers’ attention without rambling on.

Consider your audience: The voice and tone you choose for your biography largely depends on your audience and personal goals. If you’re looking for a job and are writing primarily for recruiters, you’ll want to use a serious, professional tone. On the other hand, if you’re creating an Instagram bio , consider using more casual, conversational language that reflects your personality.

Add humor: Relatedly, consider adding humor when appropriate. This is especially valuable if you’ve founded your own business or created your own website , as it can give you a distinct brand identity while helping your audience build a stronger sense of connection with your brand.

Link to your website: When writing a bio for a platform other than your own website - a social media page, another company page, or a guest blog or publication - remember to include a link to your website. This will help you promote your website while highlighting your professionalism and authority.

Adapt for different platforms: You’ll most likely need to adapt the length and writing style of your biography to suit different platforms. For example, you may place a longer bio on your website’s About page and a shorter one on your LinkedIn page. In these cases, use the same main principles for writing a bio while scaling down the most important elements.

By following these tips, you can create a powerful bio that helps you stand out in your field and allows your audience to get to know you better.

How to write a bio in four sentences or less

Really need to create a super short bio? We'd suggest following some of the tips above, just condensing them into less word for a short bio that still makes impact.

But if we really had to choose we'd say focus on - you, your professional role and company. That condenses everything that matters for bio into three sentences. Humor, creativity and uniqueness still all matter - just use fewer words to convey them.

Creating a bio for your website

As we’ve noted in the examples above, one of the most strategic places to put your bio is on your website - so be sure to consider it within your web design plans. Whether the goal of your site is to start and promote your business , showcase your design portfolio or display your resume, including a bio gives your audience a glimpse into the person behind your content. It can also kickstart your professional growth . Show the world what you do, how you do it and why it matters, and people will be drawn to your passion and inspired by your experience.

Pro tip: You can add a bio to many different types of websites, so using templates can help you create yours faster. For example, if you're creating a portfolio website , explore portfolio website templates to help you get started.

Creating a bio for social media

Crafting a professional bio for social media is vital as it introduces you or your brand, and it builds credibility and trust. A well-written bio establishes your expertise, attracts the right audience, and fosters engagement. It helps maintain a consistent brand image, optimizes search and discovery, and opens doors to networking and career opportunities. A compelling bio delivers a concise, informative snapshot of who you are, what you do, and the value you bring, leaving a lasting impression on visitors and potential collaborators alike.

You may need to edit your bio depending on which social media platform you plan to use it on. Some of the most popular ones include Twitter, Facebook, Instagram and TikTok. Focus on getting your bio right on the platforms you plan to focus your personal or brand social media marketing efforts on.

Writing a bio with AI

If you're looking to write your bio fast while creating your website, consider using an AI text generator to build your draft. You'll still need to make sure it goes through. an intensive editing process, so that it really captures the essence of who you are and your professional skills. A bio is about much more than just basic information, so don't forget to include the storytelling too. Build a website with Wix and you can make use of the in-built AI text generator within its Editor .

Why good bios are important for a professional

In a world where first impressions matter, a well-crafted bio can make a significant impact in establishing trust and credibility with potential clients, employers or collaborators. It also offers insight into your personality and values, helping to forge authentic connections with your audience. It acts as a powerful tool for personal branding, allowing you to differentiate yourself in a competitive landscape and leave a memorable impression.

A strong bio also serves as a gateway to opportunities, whether it's securing new clients, landing job interviews or establishing partnerships. It acts as a professional introduction, allowing you to showcase your expertise. A polished and impactful bio is essential for you to effectively communicate your professional identity and stand out in your field.

Writing a bio without experience

Writing a bio when you don't yet have experience can be challenging, but it's an opportunity to showcase your potential and aspirations.

Begin by highlighting your educational background, skills and any relevant coursework or projects you've completed. Focus on your passions, interests and personal qualities that make you unique. Consider including volunteer work, internships or extracurricular activities that demonstrate your commitment and initiative. Emphasize your eagerness to learn and grow in your chosen field and express your future goals and aspirations. Don't be afraid to be honest about your current stage and your willingness to gain experience and develop professionally.

If you don't know what to write in your bio, start by brainstorming your key experiences, achievements, skills and personal attributes. Consider what sets you apart and what you want others to know about you. Look for inspiration from other bios or profiles in your field, and consider seeking feedback from friends, mentors or colleagues. Don't hesitate to highlight your passions, interests and goals, as well as any unique experiences or perspectives you bring to the table. Remember to keep it concise and engaging, and don't be afraid to revise and refine your bio until it accurately represents you.

How to write a bio FAQ

What is a short bio.

A short bio, short for biography, is a concise summary of a your life or professional background. It provides a brief overview of your key achievements, qualifications, experiences, and relevant details. Typically written in the third person, a short bio is often used in various contexts, such as professional profiles, social media accounts, introductions for speaking engagements, author descriptions, and other situations where a brief introduction is required. The length of a short bio can vary, but it's generally kept to a few sentences or a short paragraph to provide a snapshot of the person's background and expertise.

How do I write a bio about myself?

What should i include in a short bio, how do you write a fun bio for work, how do i make my bio stand out, related posts.

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How to Write a Short Bio: Templates, Examples

By Status.net Editorial Team on June 16, 2023 — 12 minutes to read

• How to Write a Short Bio Part 1
• What to Include in a Short Professional Bio Part 2
• Example of a Formal Short Bio Part 3
• Example of a Casual Short Bio Part 4
• Examples of Well-Written Short Bios Part 5
• Short Bio: Best Templates Part 6
• Tips for Writing a Short Bio Part 7
• Optimizing Your Bio for Different Platforms Part 8

A short bio is a concise and informative summary of your professional background, accomplishments, and personal interests. It’s an opportunity for you to introduce yourself to others, whether it’s for networking, job applications, or social media profiles. By writing a short bio, you allow others to quickly understand your expertise, strengths, and personality.

As you write your short bio, consider your audience and tailor the content accordingly. You might want to have different versions of your bio for varying contexts, such as a professional conference, a job application, or a social media platform. Regardless of the situation, strive to be authentic and maintain a tone that reflects your personality while also adhering to professional standards.

Part 1 How to Write a Short Bio

When writing a short bio, first focus on being concise and relevant. A short bio should be approximately 4-6 sentences or about 150 words. Be sure to highlight your achievements, experience, and expertise with confidence and clarity.

To start, introduce yourself briefly, including your name, title, and current role or profession. Next, mention your most significant accomplishments in your field thus far. This can include awards, certifications, publications, or any other relevant milestones. Discuss your current work and projects, providing the reader with a snapshot of your professional life. Make sure to emphasize your unique strengths and specialties. Then, touch upon your education or any other credentials that showcase your expertise.

Here’s an example to follow:

“Jeremiah Smith, an award-winning graphic designer, specializes in creating visually stunning websites and marketing materials for a diverse clientele. With over 10 years of experience, Jeremiah has led branding projects for major corporations and small businesses alike, receiving accolades for his innovative design solutions. Currently, he serves as the Creative Director at X Design Studio, where he is dedicated to helping clients grow their digital presence. Jeremiah holds a Bachelor of Fine Arts in Graphic Design from the prestigious Art Institute of Chicago.”

For a stronger impact, customize your short bio by tailoring it to the specific platform, audience, or purpose. By prioritizing information and emphasizing the most relevant points, you can create a brief, engaging bio that showcases your unique skills and accomplishments.

Part 2 What to Include in a Short Professional Bio

• Your job title and current role : Start by mentioning your current role and the industry you’re working in. This helps to establish your expertise and gives readers an immediate understanding of your professional focus.
• Career accomplishments and milestones : Highlight a few significant achievements in your career thus far. These can be successful projects, promotions, or awards you’ve received. Be specific about what you’ve accomplished and how it demonstrates your expertise.
• Skills and qualifications : Briefly mention the key skills and qualifications you possess that make you an expert in your field. This can include technical abilities, soft skills, certifications, or degrees.
• Interests and personal touch : Add a few personal details that showcase your interests and passions outside of work. This can humanize your professional persona and help you connect with readers on a more personal level. However, be careful not to share too much personal information.
• LinkedIn and networking opportunities : Include a link to your LinkedIn profile or other professional social media accounts. This provides readers with an opportunity to connect with you and discover more about your background.

To present this information effectively, write your short professional bio in the third person and maintain a confident, knowledgeable, and clear tone of voice. Keep the content concise and easy to understand by breaking it into paragraphs and using formatting elements such as bullet points and bold text when necessary.

Here is one more example of a well-crafted short professional bio:

“John Smith is a seasoned marketing manager with over 10 years of experience in the tech industry. He currently leads product marketing efforts at X Company, where he has successfully launched new products and significantly increased market share.

John holds a Bachelor’s degree in Business Administration and is certified in digital marketing. His expertise includes strategic planning, content creation, and driving brand awareness through innovative campaigns.

In his free time, John enjoys hiking, photography, and volunteering at the local animal shelter. Connect with him on LinkedIn to learn more about his professional experience and accomplishments.”

Taking Into Account Personal and Professional Aspects

Try to strike a balance between your personal and professional aspects:

• Make sure to mention any relevant professional accomplishments and skills that showcase your expertise in your field. If you are a student or a working professional, add details about your university, current position, or professional experiences that give readers an insight into your capabilities.
• Don’t forget to add a touch of personality to your bio. Including personal details, interests, and hobbies will make you more relatable and create a connection with your audience. However, try to keep these personal elements brief and relevant to your overall bio. For example, if you are writing a bio for a personal website or Twitter, you could mention that you are an avid painter or a dedicated volunteer at a local animal shelter.

When writing in the second person, use short paragraphs to make your bio easy to read and understand. For instance:

• Full name: Briefly mention your full name at the beginning of your bio.
• Professional skills: List your core skills and accomplishments in bullet points or a table format.
• Personal interests: Share some hobbies or interests related to your profession or that showcase your values.
• Personal goals or mission statement: Include a sentence or two about your professional philosophy and core values to give readers a sense of your personal brand.

Related: How to Write a Personal Mission Statement (20 Examples)

Be cautious with the contact information you provide, especially if your bio will be accessible to the public on your personal website or social media profiles. Make sure only the necessary details are included to avoid any privacy concerns.

In summary, your short bio should be a reflection of both your personal and professional self. Showcase your skills and accomplishments while adding personal touches to make it engaging and relatable. Keep the text concise, use appropriate formatting, and remember to maintain a confident, knowledgeable, neutral, and clear tone throughout your bio.

Related: What Are Your Values? How to Discover Your Values

Selecting the Tone for Your Short Bio

Selecting the right tone for your short bio is crucial to portraying yourself in the way you want to be perceived. Consider the context in which the bio will be read and choose a tone accordingly. There are two main tones you can adopt: formal and casual.

Part 3 Example of a Formal Short Bio

Formal Tone : If you’re writing a bio for a professional context, such as a job, conference, or publication, opt for a formal tone. This means using more sophisticated language, avoiding slang, and maintaining a professional vibe throughout the bio. To achieve this, write in complete sentences, utilize proper grammar and punctuation, and highlight your achievements and expertise. Be sure to remain confident and clear in your writing. Example: “Dr. Jane Doe is a renowned expert in the field of molecular biology, with over 15 years of research experience to her credit. As the recipient of several prestigious awards, Dr. Doe’s groundbreaking work has had a significant impact on the scientific community.”

Part 4 Example of a Casual Short Bio

Casual Tone : A casual tone works well for less formal situations, such as bios on personal websites, blogs, or social media profiles. Here, you can use more relaxed language and showcase your personality. However, it’s still important to sound knowledgeable and approachable. Feel free to use contractions, incorporate humor, and speak directly to your audience to create an engaging tone.

“Hey there! I’m John, a travel enthusiast who loves exploring new cultures and tasting exotic dishes. When I’m not backpacking across the globe, you can find me geeking out about the latest tech gadgets or sipping on a well-crafted cocktail.”

In both cases, whether formal or casual, always ensure that your voice is confident, neutral, and clear. Remember to keep it concise, avoid exaggeration or false claims, and maintain a second-person point of view.

Part 5 Examples of Well-Written Short Bios

Short bio example 1.

Jane Smith is a marketing expert with over 10 years of experience in helping brands elevate their online presence. With a passion for storytelling, Jane excels in creating content that engages and inspires. In her free time, she enjoys hiking, photography, and exploring her city’s local coffee shops. Connect with Jane on LinkedIn or follow her on Twitter @JaneSmith.

Short Bio Example 2

John Doe is an experienced software engineer with a knack for developing cutting-edge applications. Specializing in full-stack web development, John’s expertise lies in JavaScript, Python, and Node.js. When he’s not coding, John can be found playing the guitar, tutoring local students in programming, or cheering on his favorite esports team.

Part 6 Short Bio: Best Templates

Short bio template 1.

[Your Name] is a [industry or profession] expert with [number of years] of experience in [specific skills or areas of expertise]. [He/She/They] specializes in [technical skills or industry knowledge] and has a passion for [relevant interests]. In [his/her/their] free time, [your name] enjoys [hobbies or activities]. Connect with [your name] on [social media platforms] or through [his/her/their] website.

Short Bio Template 2

As a [occupation or field], [Your Name] incorporates [unique qualities or skills] to produce [specific type of work]. With a background in [relevant experience], [He/She/They] has been able to [achievement or accomplishment] through [personal path or passion]. When not [working or creating], [Your Name] spends [his/her/their] time [hobbies or activities], always seeking new inspiration.

[Your name] is a [profession or role] with a background in [relevant expertise or industry]. [He/She/They] earned a [degree] in [field] from [institution]. [Your name] has [number of years] experience in [profession/industry], providing [valuable service or skill]. Outside of work, [your name] enjoys [hobbies or personal interests]. Connect with [your name] on [social media platform] or visit [your website or portfolio].

Customize these examples and templates to fit your own unique skills, experiences, and personality. Using a second person point of view, focus on the key aspects you want your audience to know about. Be confident and transparent about your achievements and interests, and let your short bio speak for itself. Happy writing!

Part 7 Tips for Writing a Short Bio

• Know your target audience : Consider the people who will be reading your bio and focus on the information that will be most relevant to them. Tailor your bio to best serve their needs and expectations.
• Highlight your accomplishments : Share information on your achievements, awards, and notable experiences. This will give your audience an understanding of your expertise and success in your field.
• Include your goals and mission statement : Tell your audience what drives you and what you hope to achieve. This can help create a connection with the reader and showcase your dedication to your work.
• Maintain a professional tone : Write in a clear and concise manner, avoiding casual language and slang. A confident and knowledgeable tone will convey your competence in your field.
• Keep personal information to a minimum : While you may choose to mention some personal tidbits, be mindful of what you share. Focus on information that enhances your professional image, rather than oversharing personal details.
• Promote your brand and company : If you represent a business or have a personal brand, mention your company name and mission statement. This can help reinforce your brand identity and make a stronger impression on your audience.
• Prioritize transparency and authenticity : Be honest about your experience and qualifications. Avoid exaggerating or making false claims in order to maintain trust with your audience.
• Limit self-promotion : While it’s important to show off your accomplishments, be sure to keep the focus on meaningful information rather than excessive self-promotion. This will help engage readers and build credibility.
• Use formatting to enhance readability : Break up your bio into paragraphs, use bullet points for lists, and bold text for important details. This will make it easier for your audience to read and understand your bio.
• Include contact information : Provide a way for your audience to get in touch with you, whether it’s an email address, phone number, or a link to your website.

Part 8 Optimizing Your Bio for Different Platforms

On LinkedIn , focus on your professional achievements and skills. Use bullet points or a table to highlight your most significant accomplishments. Feel free to include any relevant certifications, courses, or awards. Remember that LinkedIn is a professional networking platform, so maintaining a professional tone is crucial.

For a resume , your bio should be concise and focus on summarizing your career history and specific expertise. Make it easy for potential employers to grasp your main strengths quickly. Use bold text to emphasize crucial information, such as your job title, years of experience, or industry-specific skills.

On a personal website , you have more freedom to express your personality and showcase unique aspects of your life. Consider adding anecdotes, hobbies, or personal achievements to give visitors a glimpse of who you are outside of your professional life. You can also touch on your professional capabilities but keep it concise.

For Twitter , keep in mind the character limit for bios and make every word count. Capture your profession or industry, and maybe add a touch of your personality or interests through emojis or hashtags. It’s common to see authors and celebrities mention their latest projects, books, or achievements here.

Frequently Asked Questions

What are the essential elements of a short bio.

A short bio should include:

• Your name and current role or profession.
• Brief background information including education and relevant work experience.
• Notable accomplishments or skills relevant to your profession.
• Personal interests or ambitions that showcase your personality.
• A call-to-action, such as directing readers to your portfolio or LinkedIn profile.

How can I create a compelling short professional bio?

To create a compelling short professional bio, follow these steps:

• Start strong with a clear and concise introduction.
• Focus on your most relevant qualifications and experience.
• Highlight key achievements and successes.
• Provide a personal touch that showcases your unique attributes.
• Keep it brief and easy to read, aiming for around 100-150 words.

What are some tips to make my short bio stand out?

• Use vivid language and strong, active verbs.
• Tailor your bio to your audience, emphasizing information that is most relevant to them.
• Share a unique or unexpected personal interest to pique interest.
• Edit and proofread your bio carefully, ensuring it is free of errors and reads smoothly.

How can I tailor my short bio to different contexts?

Adjust your short bio for different contexts by:

• Focusing on relevant skills, experience, or accomplishments for each specific audience.
• Adjusting the tone or language to suit the platform (e.g., more casual for a social media profile or more formal for a conference bio).
• Emphasizing specific personal interests or accomplishments that align with the context or audience.
• Updating your call-to-action as needed to direct readers to relevant content or profiles.

Related: 150+ Awesome Examples of Personal Values

• How to Write a Personal Mission Statement (20 Examples)
• How to Live By Your Values

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Standout Professional Bio Examples (And Why You Need One)

• Compelling examples of bios in multiple contexts

The difference between a resume and a professional bio

Who needs a professional bio.

A professional bio is one of the most important pieces of writing you have in your toolkit.

What career and business opportunities will yours open up?

Effective examples of bios in multiple contexts

When crafting a professional bio, be intentional about the content you include and tailor it according to your objectives.

You want to adjust it accordingly based on where it appears and what type of organization and industry you work for.

The purpose of a professional bio is generally to:

• State who you are and what you do
• Provide examples of your work experience that bolster your credibility
• Tell your story

How you go about accomplishing these three goals, however, will vary.

We compiled a variety of professional biographies and explain why they are effective to give you an idea of how to structure yours.

Company Website

If you are a senior executive, you may have a professional bio on your company’s website: this is the most common request we receive.

John Hartman | Equifax

John Hartman put together a classic example of an executive bio for the Equifax website.

His writing is clear, concise, and follows the standard structure for an effective bio by providing:

• Name and current role at Equifax
• Previous experience at Equifax
• Early career history
• Education summary

If you’re looking for a conventional executive bio, you’ll want to follow this format.

It’s perfectly acceptable to use the bio you have on your company’s website for LinkedIn.

However, don’t forget that LinkedIn is still a social media platform, so it’s okay to infuse a bit more personality into it !

Jay Shetty | Personal Brand

Jay Shetty's profile headline and introduction quickly grab the reader's attention: “Meet the former monk who is making wisdom go viral.”

He further builds his credibility by explaining that he has 200 million views on social media, over one million followers, and was selected for Forbes 30 Under 30.

His bio also includes information about his education and career history while infusing elements of his story (we’ll forgive him for grammar errors and focus on content – because it is quite strong).

At the end, he also provides a preferred method of contact.

You should consider including your email address or a link to your LinkedIn profile as well – especially if your bio doubles as a marketing tool.

Need help crafting a compelling executive bio? Learn more  about our professional bio writing service!

Personal Website

We’re increasingly seeing freelancers and even professionals in the corporate sector building personal websites that serve as an online resume, so to speak.

If you are a freelancer or run your own business, think carefully about your copy and what kind of message you are sending to your readers.

• What kind of impression will they have of you and your business after reading your bio?
• Does your bio leave them inspired and wanting to talk to you further?
• What is the image you are trying to convey? Do you want to come off as polished and professional, engaging, fun, or simply vanilla? 

Melanie Everett | melanieeverettco.com

Melanie Everett runs an independent real estate firm in Chicago.

Coming from a journalism background, she has leveraged her writing skills to drive her real estate business. She currently ranks in the top 1% of real estate agents in Chicago.

Conference or Event Pamphlet

If you are speaking on a panel or delivering a keynote speech, you may have the opportunity to provide a bio to be featured in the event’s print materials.

For these types of communications, you’ll want your bio to be shorter than what you might include on a company page or LinkedIn summary.

These executive bios build credibility for you and your company while positioning you as an expert on the topic you will be speaking about.

Vera Manoukian | Hilton

Hilton’s Vera Manoukian provided this bio when she spoke on a panel titled “The Rebirth of Full-Service Hotels,” effectively demonstrating her leadership experience in the hospitality sector.

Nonprofit Bio

Executive bios for nonprofit organizations often employ a storytelling approach to introduce readers to the organization’s mission and impact.

Ben Justus | EGBOK Mission

In his bio, Ben explains why he started EGBOK, the work his organization does, and how it has grown.

When you contribute to an industry blog, you may have the opportunity to include additional information that showcases why you are an authority on the topic.

In this context, your bio can also serve as publicity for your company.

Arianna Huffington | THRIVE Global

Brian X. Chen | The New York Times

Other times, you may have very limited space to work with, making it especially important to consider how each word is adding value.

A resume and professional bio are both personal marketing documents, but they are used in different contexts.

Resumes are generally used to apply for a job. They are written in third person, follow a conventional structure, and are predominantly based on facts and achievements .

Professional bios , on the other hand, provide a more informal narrative that serves as a compelling elevator pitch.

Professionals of all experience levels can benefit from a bio.

As an executive, you should have a long and short version of a professional bio to use on:

• Corporate or personal websites
• Company marketing brochures
• Conference materials

If you are a student , you can use yours as a LinkedIn summary or on the webpage of a campus organization you are a part of. You never know who might come across it!

We hope these examples have given you inspiration and an understanding of what to include in your bio.

When writing yours, remember to keep the following in mind:

The best bios are written thoughtfully and intentionally, so it’s not something you want to leave to the last minute!

Need a compelling executive bio? You can order our professional bio writing service here !

About resume pilots.

Resume Pilots is an award-winning executive resume writing firm and a proud member of the Professional Association of Resume Writers and Career Coaches. Our previous clients include CEOs and senior executives at the world's leading companies.

As a professional services firm, we take your reputation seriously. We are committed to delivering writing excellence and superior service while operating with integrity and discretion. Recruitment firms we partner with also trust us to consistently deliver quality documents for their clients.

Our writers have studied in the Ivy League and other top-tier universities and have strong writing backgrounds coupled with industry experience.

Here's how we can help you:

Resume, Cover Letter, and LinkedIn Writing Services : If you are looking for end-to-end support, hire one of our professional resume writers to rewrite your documents from the ground up.

Executive Resume Template Downloads : If you plan to prepare your own resume, consider using one of our classic, ATS-friendly resume templates for Microsoft Word.

To learn more about our services, book an introductory call with our founder here or email [email protected] .

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About the Author Matt Glodz

Matt Glodz is the Founder and Managing Partner of Resume Pilots and a Certified Professional Resume Writer. After studying business communication at Cornell University, Matt worked within Fortune 500 companies, where he noted that qualified candidates were frequently denied interview opportunities due to poorly written documents. At Resume Pilots, Matt combines his business and writing background - which includes prior work for a Chicago Tribune publication - to craft resumes that give his clients the best chance of landing interviews. He works with clients ranging from CEOs to recent graduates and has been writing resumes for over 10 years. He has been quoted on numerous business and career-related topics in outlets including Business Insider, CNBC, Fortune, Glassdoor, The Ladders, and Thrive Global.

Related Articles

Should you include a professional summary on your resume, how to write a strong linkedin summary, when should i remove internships from my resume.

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Enchanting Marketing

Writing advice for small business

How to Make a Rip-Roaring First Impression, With Only a Few Words

by Henneke | 95 enchanting opinions, add yours? :)

Even if you don’t need a short bio right now, writing one is a great exercise. You learn how to say more with fewer words. You learn how to position yourself and how to make a big impression from the get go.

But last week, when I started preparing this post, I made a big mistake.

I looked for inspiration on Twitter.

I scrolled through hundreds of Twitter bios, and I almost gave up on this post because most Twitter bios are utterly dull. And they say almost nothing, like this:

If you try to explain a lot about yourself in a few words, you end up saying nothing. No personality. No big impression. No sense of connection. You sound the same as everyone else.

So, I decided to look for inspiration elsewhere. How do fiction writers introduce their characters in a few words?

4 examples of saying a lot with a few words

I skimmed through the books I’ve read recently to look for powerful descriptions of people.

For instance, here’s how William Kent Krueger typifies an FBI agent in “Boundary Waters:”

How powerful is that comparison to a pit bull! In only 11 words, Krueger gives us a picture of an aggressive guy who’s persistent, too.

It made me wonder, if I were a dog, what dog would I be?

Here’s a longer example by the same author, from “Iron Lake:”

Why is this description powerful? Because the author focuses on one aspect of the person and he gives specific examples. The description is visual, so we can picture this guy sparking fires, tying knots and shooting arrows.

The description may be a little long, but you can cut it easily by 50% and still make a strong first impression.

Here’s a shorter character description from “Force of Nature” by Jane Harper:

Three specific details (jogging, yoga, deep-conditioning her hair) help us visualize Breanne, and we can imagine how she glows even in the unflattering office light.

And here’s one last example, from “The Keeper of Lost Things” by Ruth Hogan:

Again, three specific details express a lot: Prozac, Pinot Grigio, and pretending things weren’t happening. The words even alliterate for extra flair.

When you focus on one character trait or skill, you make a strong first impression and you invite readers to learn more. In contrast, if you try to say too much too quickly, a description of a person falls flat and nobody gets a feel of who they really are.

Choose what to tell in your professional bio

Of course, the fiction examples above are different—you wouldn’t write about Prozac in a professional bio.

But the principles of what makes a good bio remain the same. Choose what to tell and, even more importantly, what to leave out. You can’t cram your whole resume into one sentence. You have to be ruthless.

Twitter bios, for instance, are so short, you can only communicate one or two things. That’s it.

The Twitter bio of Unbounce’s Twitter bio explains what their software can do for you plus invites you to try it for free:

Casa Collective’ s bio is similar:

If you Tweet as a person rather than a company, I like bios that show personality, like this one from Haemin Sunim :

Or this bio from John Espirian :

Or this bio from Kitty Kilian is one of my favorites (the original is in Dutch, I’ve translated it):

A Twitter bio is super short, and you can only make a strong impression if you choose carefully what to write.

The 3-step professional bio

If you have three sentences, for instance for an author bio, then you can say a little more. But be careful, the same rule applies as with the one-sentence Twitter bio. Keep it short to make a strong first impression.

For a three-sentence bio, consider these three objectives:

• Tell readers who you are and what you do
• Reveal a glimpse of your personality
• Encourage readers to find out more

For instance, my author bio is:

In three sentences, I explain what I do (writing), what I’m passionate about (stamping out gobbledygook), and I invite you to join my course so you can learn how to write more persuasively.

Jessica Blanchard’s bio follows a similar pattern:

And Ry Schwartz’ bio:

Ry’s call to action isn’t very strong, but “More reclusive than J D Salinger on a rainy day” is a superb way to sketch a personality in a few words.

How to write a short professional bio

Want to make a big impression with only a few words?

Communicate less instead of more:

• Start with answering the question “What do you do for your clients?” or even better, finish this sentence suggestion from Mark Schaefer : “Only I …”
• Think about an adjective describing you well; Ry Schwartz uses “reclusive,” John Espirian uses “relentlessly helpful” and I use “irreverent” to present a quick glimpse of who we are
• Consider a metaphor to communicate more with fewer words, such as the “pit bull” or “delicate as a hand grenade”
• If you have space, have a clear call to action

In short, communicate what makes you different, and tell clients what you can do for them.

Because a professional bio is not only about you.

It’s about your clients, too.

Books mentioned in this post:

Recommended reading on writing about yourself:

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Reader Interactions

Leave a comment and join the conversation cancel reply.

October 31, 2022 at 6:47 am

I love the way you gave perspective from books you read. Very relative and helpful. You keep giving me hope. Thanks.

October 31, 2022 at 11:56 am

Thank you, Andrea. Happy writing!

April 12, 2022 at 4:47 pm

Thank you! You helped me make mince-meat out of a project I always dread – writing about myself. I did a quick search for “how-to write a short bio” and found my favorite writing coach.

April 12, 2022 at 4:52 pm

Wow. So, great when Google delivers exactly what you need 🙂

And thank you also for your lovely compliment, Amy.

April 28, 2021 at 6:50 am

Yet another comment, after reading close to a couple of your blogs. l was asked for a CV today.

Definitely l had to make a search. These days whether its something about writing or not, l first input the search topic with Enchanting marketing.

Sometimes , l don’t really get anything but when l do like today, l get so thrilled like a 5 yr old with a favourite toy.

Let me stop gaping at your writing talent and get on with this CV. Thank you & Cheers Henneke.

April 28, 2021 at 3:14 pm

Thank you so much for your lovely comment, Emily. I’m happy to read that Enchanting Marketing has become your #1 source for writing advice. Good luck with your CV!

April 18, 2021 at 2:34 am

I just landed a new job and it asked or a bio. Google search presented me this page. What an inspiration… I am awestruck with all the wisdom in such a simple article, yet as mentioned in several of the conversations below, writing something in a couple of sentences is far from easy. I admire your patience in responding to every individual comments. I have to get to work in crafting something, which is creative and succinct.

April 18, 2021 at 2:40 pm

I’d say clarity is more important for a bio than being creative. Being creative is a bonus but not a requirement. Have fun!

September 17, 2020 at 4:17 pm

Thanks so kindly, Henneke. This is quite helpful. How does this sound? …a necessity entrepreneur, an avid supporter of women entrepreneurs, helping small business owners fall in love with their business.

September 17, 2020 at 6:38 pm

I don’t know what a necessity entrepreneur is. But maybe your potential clients know what it means? That’s more important than that I understand it.

Also, it’s unclear whether you help women entrepreneurs or all small business owners. Better to focus on one option instead of risking confusion.

May 23, 2020 at 2:17 pm

If you are guest posting, and the host allows for a much larger word count, do you include more information about yourself? Or do you include more about your results? Or maybe 50/50? Or do you keep with the short version and resist the temptation to change it any? I’ve revisited all mine and realize how many I’ve written, each one different. I did not even notice until now, so thanks for that! Also, I totally missed this post when it first came out and I love the tulips! 🙂

May 25, 2020 at 5:54 pm

I’m glad you enjoyed the tulips!

The bio I used for guest posts is the one I’ve quoted above—it’s relatively short: Henneke Duistermaat is an irreverent writer on a mission to stamp out gobbledygook. Get your free 16-Part Snackable Writing Course for Busy People and learn how to enchant your readers and win more business.

Even if the host allowed a longer word count, I haven’t made the bio longer because I was always afraid people might lose interest. Also, they’ve already read a whole blog post so I’ve already built up some credibility with the advice shared in the blog post. But this is not something I’ve tested. It’s mainly based on gut feeling.

May 26, 2020 at 2:20 pm

Thanks! I think you’re right!

January 24, 2020 at 7:46 am

Am still kind of confused but am sure I will get the hang of it. And thanks a lot.

January 24, 2020 at 10:54 am

What are you confused about?

January 9, 2020 at 1:28 am

Wow! I stumbled upon this article and you have successfully provided the unique kind of ideas I was looking for : ) Thank you Henneke

January 9, 2020 at 7:17 pm

Great 🙂 That makes me happy. Have fun writing your bio!

November 29, 2019 at 5:40 am

Oh my goodness! marvellous bio! Thanks.

Thank you so much, Henneke.

November 29, 2019 at 10:02 am

Thank you, Abhinav. Happy writing!

November 12, 2019 at 3:01 pm

You play a dangerous game when you automatically assume the author who used the pit bull reference intended to self describe as aggressive. Pit bulls are smart, loyal, and most of all affectionate. People like you spread bullshit and add fuel to the stereotype of pit bulls being aggressive. I highly suggest you educate yourself before making a blanket statement about an entire breed of animal. Do better.

November 13, 2019 at 12:24 pm

The example you refer to is from the book Boundary Waters, a thriller, in which the author describes one of the characters as a pit bull. He’s an FBI agent who comes as aggressive in the remainder of the book—a fighter.

April 2, 2019 at 8:06 pm

Hey, Henneke

Really amazing content. You’re right a bio define a person and about their profession. So, it’s really important to write an awesome bio.

I will surely use your tips to write a bio of my social media accounts.

March 11, 2019 at 4:15 am

Marketer ExtraOrdinaire, like a stick of dynamite, I blow up old ways of doing things, bringing fresh ideas. The pink bunny can’t keep up with me.

September 13, 2018 at 2:21 pm

Indeed, First impression last. Making an impressive bio needs to convey both professional status and personality in just one or two sentences which creates a great impact to the clients. I sometimes say or write too much and with no idea where I was going or how to make the points I wanted to make. It felt like I was writing in circles, as a result, I was saying nothing. Now I know what to do. Great tips, Henneke.

September 16, 2018 at 1:10 pm

I sometimes write in circles, too, as I don’t always know exactly what I want to say. The writing (even when it seems to lack purpose at first) somehow brings clarity.

Thank you for stopping by to leave a comment, Rebecca 🙂

September 4, 2018 at 11:12 am

September 4, 2018 at 11:33 am

My pleasure 🙂

September 1, 2018 at 10:08 am

I like this. Always playing around with my Short bio for social platforms. What do you think of my twitter bio? @davidgcant Could be improved? Give it your best shot..

A risk savvy health and safety consultant extraordinaire. Cuts through the red tape (with a safety blade of course).

September 2, 2018 at 10:09 am

I like it! I’d be tempted to shorten it a little: A health and safety consultant who cuts through the red tape (with a safety blade of course).

(To me the phrase “risk savvy” doesn’t add a lot as it feels kind of obvious that a health and safety consultant would be risk savvy; that’s what the job is about.)

September 2, 2018 at 4:04 pm

That reads much better. I was thinking of replacing risk savvy with refreshingly practical. What do you think? Or is that too much?

September 2, 2018 at 7:06 pm

The idea of cutting through the red tape already indicates that you’re a practical person rather than a rigid you-must-follow-the-health-and-safety-bible type of person. So, I’d say adding “refreshingly practical” doesn’t really add anything new as you’re already telling me you’re practical—you understand client’s objections to health and safety, plus you have a sense of humor.

August 30, 2018 at 11:44 pm

Succinct and thereby excellent, Henneke. Have to say that I’ve read words of many copywriters down through the years – some good, some not so, even if near-legendary. In my view your writing surpasses most. Please keep it up because I’ve learned more from your posts than I have from anyone else’s. Thanks.

August 31, 2018 at 6:27 pm

Thank you for your lovely compliment, Paul. I appreciate it! 🙂

August 25, 2018 at 5:37 am

Helpful post, as always, but I really just wanted to say I love that drawing of Henrietta with the tulips, on a grey cold day like this it warms the brain. Not that I can complain about the greyness, NSW where I live is 100% in drought. I hope the clouds are not just teasing. Best wishes.

August 26, 2018 at 6:09 pm

Thank you for your lovely compliment on my drawing, Susan. That means a lot to me! I hope the rain will soon ease the drought.

August 22, 2018 at 11:16 pm

Hi Henneke, Thank you for a great article. I remember when I first came across your website (last year, I think), I was impressed with your bio and made a note to myself to use that as inspiration for mine. Of course that mental note was long forgotten! This time I’ve already made a first attempt for my social media profiles. I’ve been a longtime lurker, this is my first comment and is well overdue. Thank you, I can hands down say I learn and apply something useful from each of your articles.

August 23, 2018 at 9:27 pm

That is a lovely compliment. Thank you so much, Sonna.

My own default position is also as a lurker. I appreciate you stepped out of the shadows to leave a comment. Have fun with writing your bio! 🙂

August 22, 2018 at 5:19 pm

Insightful and incisive Love the bonus recommended reading 😉

August 22, 2018 at 5:59 pm

Thank you, Hendel. I started adding the bonus reading recommendations recently as I was asked so often 😉

August 22, 2018 at 9:54 pm

Henneke: Few weeks back, I came across your blog and book.. I not only like the content and but also the presentation style… I really enjoyed reading this article and response to the many comments. I drafted by first short Bio.. could you please review.

Chandra is a Meditation teacher who conducts group meditation sessions in-person and on-line. Her Smile and her energy is contagious. Her mission is to encourage many human beings e discover their SacredSpace with in them through powerful meditations. To know more about her journey visiti https://cvelpula.wixsite.com/mysite/blog/journey-of-discover-sacredspace-within-you

August 23, 2018 at 9:05 pm

I’m glad you enjoyed this blog post, Chandra.

Great start on your bio! You may want to consider tightening it a little as you use the phrase “meditation” three times which is usually a sign that you can make it more concise. Also, what is the ultimate aim of your clients? Do they want to discover their SacredSpace or is their a deeper purpose? This depends on who you’re targeting.

August 22, 2018 at 4:07 pm

Henneke, this was very helpful. I always have trouble writing shorter than longer. Thanks.

August 22, 2018 at 4:19 pm

I find it hard, too. And it takes more time than writing longer!

Thank you for stopping by, Anita. It’s always good to see you 🙂

August 22, 2018 at 11:38 am

Now this is powerful stuff Henneke and thanks to Lisa for sharing.

My bio is not more than 2 sentences but this post just gives me a lot of meat.

I hope you are having a great week

August 22, 2018 at 1:46 pm

I’m glad you found it useful, Enstine! Thank you for stopping by.

August 22, 2018 at 11:12 am

Hi Henneke – I hadn’t thought about writing my bio until I read your article. I’ll certainly follow your excellent advice when the time comes. All the best from sunny Sydney

I hope you’ll have fun writing your bio, Paul!

Still cloudy here 🙂

August 22, 2018 at 1:43 am

Ooh! Henneke, I love this post. And who is Henrietta’s new friend? (or did I miss a post?) I love these smart, smooth, silky, bio’s and would love to add my own. I dream♡ This is my first attempt. Your comments would be sooooo valued. Thanks for all your gorgeous posts Henneke.

Like Columbo shelling his morning egg Joan reveals you to yourself; then helps you delete what no longer serves you and install what does. Joan is an NLP Master Practitioner and Professional Coach working with coaches and service professionals. …… this would be followed with a short CTA

August 22, 2018 at 1:43 pm

Henrietta’s puppy is called Arthur. He’s been around for ages, but often I’m too lazy to draw him! 🙂

You can probably take one step further with your bio, by defining the real benefit of what you do. You can find the real benefit by finishing the sentence: she helps you delete what no longer serves you and install what does, so ….. What is it your clients really want to achieve?

August 23, 2018 at 2:20 am

Thanks, Henneke, that helps. I did add that initially but thought it made it too long – I wrote – “so that you can make a difference and make money as you grow your Lifestyle-Biz.”

I’ll keep playing with it. Now I’m thinking the Columbo piece sounds a bit ‘cheesy’.

August 23, 2018 at 9:33 pm

I’m not sure the Columbo reference adds a lot. I do like the phrase “reveal you to yourself.” I also like “make money as you grow your Lifestyle-Biz” as it’s a true benefit and it’s also specific to your audience. “Making a difference” is a little abstract. So, I’d be tempted to leave that out, or to make it more specific (but that might make it even longer).

August 23, 2018 at 11:26 pm

Terrific, just leave that Columbo piece out. I’ll play with the rest of it. Thanks for the extra input Henneke. ♡

August 21, 2018 at 11:28 pm

The short takes are never easy. How to sum up Hetty in 3 sentenceso ha! So…. Easy going until taken for granted. Lover of stationery, determined to be a success in business. Express your thoughts in our Ankara Fabric Covered Notebooks

August 22, 2018 at 1:41 pm

Great start on your bio, Hetty. It makes me wonder what’s special about your notebooks and why are you passionate about them?

August 21, 2018 at 6:14 pm

Brilliant and enchanting as usual! Thanks for confirming how difficult it can be to engagingly summarize one’s life in two sentences. I get to put a short bio on most of the articles I write for magazines and I am so disappointed in the various versions I have come up with over the years. Now I see why, I conveniently left out my real personality. Shoot.

August 21, 2018 at 8:58 pm

Don’t beat yourself up over it, Thea. Most people leave their personality out of their bios. It IS a tough challenge.

Thank you for your lovely comment. I appreciate it! 🙂

August 21, 2018 at 5:33 pm

To be honest, I skimmed your initial email the first time and, because my day is the usual crazy, didn’t click through initially. But I’m so glad I made the time to do it now. This was an absolutely genius post—and the examples were outstanding! I’ve added a note to do some brainstorming on my bio next week. As always, thank you for the inspiration and the easily digestible and actionable advice!

August 21, 2018 at 8:56 pm

You know, it really humbles me when you make time on a crazy busy day to not only read my post but also leave a comment. You’re a star. Thank you 🙂

August 21, 2018 at 4:41 pm

Thank you for the valuable insights.

August 21, 2018 at 4:47 pm

Thank you, Mohsin. Was there anything in particular you found valuable?

August 21, 2018 at 3:43 pm

Hi Henneke:

For me the fight to find the perfect bio is unending…seems like I’m forever tweaking. Also, in my constant state of evolving (or is it experimenting?) I’ve taken to changing my bio for different places I publish! Oh Henneke, why can’t I just do one perfect one like you and it’s everywhere!!! Geez. P.S. But, don’t forget you need to add “illustrator” to yours!!! Bi Oh My!! Take care, Sue-Ann

August 21, 2018 at 4:43 pm

I don’t have one perfect bio either. I don’t think the perfect bio exist. Unless you’re Andy Murray whose Twitter bio simply says: “I play tennis.” How brilliant is that? Unfortunately, that doesn’t work for mere mortals like you and me.

And you see … you already mention “illustrator,” but somehow it didn’t feel right. Also, I have a different bio on Instagram because I mainly share my drawings there. My Insta bio is: I play with words and colored pencils.

Perfection doesn’t exist. Experimentation is what life is about, isn’t it?

August 22, 2018 at 8:34 am

Ha ha! I always laugh at Murray’s twitter bio as it’s so deceptively simple yet at the same time understated. I like how he doesn’t feel the need to list out all his achievements. By the way, I missed my flight home from NYC in 2012 to see him win the US Open, which was amazing.

Anyway, back on topic, trying to do a short About section for my new “personal branding photography” landing page. Trying to work in something like my “hobbit-like stature and tenacity”, along with being eco-friendly and helping businesses save time by creating images for their online content. It’s still a work in progress.

August 22, 2018 at 1:45 pm

Murray’s bio is brilliant. I love how understated it is. And lucky you for seeing him play (and win!) live!

I like “hobbit-like tenacity” 🙂

August 21, 2018 at 3:02 pm

Thanks, Henneke. I’m honoured to get a mention here – much appreciated!

I hate it when all those letters, spaces and punctuation marks don’t amount to anything meaningful.

August 21, 2018 at 4:51 pm

It was rather demotivating to read through all those Twitter bios!

Thank you for stopping by, John.

August 21, 2018 at 2:33 pm

Great post and I loved the examples you gave.

I find writing bios very hard. I have this instinct to look at what others in my field are writing and try to find something that would work for me. It’s insane! I know it’s wrong, I know it’s useless and yet, I’m attracted to it.

What’s with the copycatting? I have a strong identity, I know what I want and who I am. Why does it all go to smoke when writing things like bios on social media?

I’m going to get to work in a dark room with no phones or internet and write away. Well, scratch the dark room part. You know what I mean. 😀

August 21, 2018 at 4:38 pm

I think most of us look at what others in our niche are doing, and there’s nothing wrong with trying to learn from others. I’ve found it’s often easier to look outside our own niche for inspiration.

And out of all the different writing tasks, writing a bio is probably one of the most difficult. Play for a while, then let it simmer for a while, and then play a little more. Have fun!

Thank you for stopping by, Elfin!

August 21, 2018 at 2:16 pm

You’re inspired me! Awesome post – and glad you said it took weeks to come up with your bio. These examples make it look easy.

August 21, 2018 at 4:36 pm

Writing so often looks easy, but we don’t see how much effort has gone into it. For me, the biggest challenge was to decide what I wanted to say. I was just starting out and I hadn’t figured out my positioning yet.

Thank you for stopping by, Cathy. I appreciate it 🙂

August 21, 2018 at 2:08 pm

Great advice Henneke! I took notes and will check out some of your examples!

August 21, 2018 at 4:34 pm

Yay! Happy bio writing, Kim 🙂

August 21, 2018 at 1:59 pm

Hi Henneke,

Thank you for this post. It came just in time as I am currently trying to re-do my website about page and social profiles.

Here’s what I came up with after reading your post. What do you think? Storytelling is my superpower, so I thought to highlight that. So how’s this for a try:

Enamored with writing. I spin engaging stories about your brand so your clients don’t have to shop elsewhere. Just call me your Fairy Wordmother.

August 21, 2018 at 4:45 pm

I’m glad this post came at the right time, Zika 🙂

Using a positive is often stronger rather than a negative. So, for instance, instead of “so your clients don’t have to shop elsewhere,” you could have: “so your clients fall in love with your brand.”

August 21, 2018 at 4:57 pm

Thank you so so much, Henneke.

August 21, 2018 at 1:38 pm

I read your post not for my own bio actually but because I need help writing one and two sentence introductions for 40 – 50 historical puzzle pages. And it works for that! Where yesterday I was plodding through it like an elephant in quick sand this morning I’m inspired to have more fun with my project. Especially love the suggestion to use metaphors. That would make a whole blog on its own. Maybe you already have?

August 21, 2018 at 4:33 pm

Yay! I’m so glad you feel inspired, Patty 🙂

I wrote about metaphors here and here . But I could probably write another one 🙂

August 21, 2018 at 1:37 pm

Brilliant and not boring bios. That draw you in.

Excellent article Henneke.

August 21, 2018 at 4:31 pm

Thank you, Ray. I appreciate your comment 🙂

August 21, 2018 at 1:26 pm

I’ve rewritten my Twitter bio using this post as inspiration! It originally said;

#Blogger & #copywriter for #software & #tech (esp #IoT). Loves #design, productivity & mindfulness. Newcastle upon Tyne. Ravenclaw. INTP. Chocoholic.

I was clearly so very guilty of trying to say as much as possible in a short space of time. Now it says this;

Chocoholic copywriter LJ enjoys making tech sound fun, not scary. She’s also as subtle as a sledgehammer and wants to help startups build brilliant blogs. Start here; http://eepurl.com/cS8g99

My dad is the one who describes me as being as subtle as a sledgehammer 😉

I love the changes you’ve made! 🙂

I wonder whether you can connect “subtle as a sledgehammer” somehow to building blogs. Something like: Subtle as a sledgehammer, she helps startups build blogs that rip through online noise. Maybe not quite, but it’d be great if you could show the advantage of being like a sledgehammer 🙂

What do you think?

August 21, 2018 at 1:23 pm

One of your best Henneke!

I’ve long felt the same about “elevator speeches” — the reason they don’t work is we try to jam too much information into what should be a single thought-provoking nugget.

August 21, 2018 at 4:27 pm

Yep, I agree with you. The idea of elevator speeches makes me roll my eyes. But that’s silly because a good elevator speech is fab. As you say, you need just one thought-provoking nugget, something to connect with your audience.

August 21, 2018 at 1:16 pm

Great tips and examples Henneke!! It’s awesome to see examples that are different from the norm. Love the visuals.

You’ve inspired me to improve my byline. What do you reckon?

Cynthia is a copywriter powered by chai lattes, rockclimbing, and remedial massages. She’s on a mission to draw out personable, reputable, small health and fitness businesses from the shadows. Grab her free guide on how to sound intelligent by writing in plain English.

August 21, 2018 at 4:25 pm

I like it. You can probably still improve the second sentence a little more. Try to find a stronger phrase than “draw out from the shadows”—something that sounds more positive, and reduce the number of adjectives as “personable, reputable, small health and fitness businesses” is quite a mouthful.

I love the idea of a free guide on sounding more intelligent 🙂

September 11, 2018 at 5:38 am

Thanks for the feedback. I find it tough to keep it simple sometimes! 🙂

How about this version? Cynthia is a copywriter powered by chai lattes, rockclimbing, and remedial massages. She’s on a mission to help small health and fitness businesses connect with clients through writing that sticks in their reader’s minds. Grab her free guide on how to sound intelligent by writing in plain English.

September 12, 2018 at 2:09 pm

Yes, I like it. If you like, you can consider making your mission more specific to your audience. For instance: help small health and fitness businesses inspire their clients to lead a more healthy lifestyle.

August 22, 2018 at 1:53 am

I like it! And Henneke’s suggestions.

August 21, 2018 at 12:27 pm

Hi Henneke, Another great of your posts here. The goal of every writer is to have their target audience read through to the last word. That said, it is genius to be artistic if a writer has to stand out of the crowd. This is where you have touched best ~ Use of a BIO ~ and a captivating bio for that matter. Most of the time readers will look at the bio of an author before they could dedicate their time to read the author’s article. You nailed it as always. I always read your articles to enrich my knowledge Keep doing what you do BEST 🙂

August 21, 2018 at 12:43 pm

Thank you for your lovely comment, Tony. A dash of creativity can definitely make a bio more captivating.

August 21, 2018 at 12:23 pm

‘More reclusive than J D Salinger on a rainy day,’ >> yes! Jealous of that.

And thanks for including me 😉 Interesting that the second sentence translates from 6 words in Dutch to 11 in English – usually it’s the other way round.

August 21, 2018 at 12:42 pm

Yes, I found it also weird that I ended up with more words. I somehow couldn’t make it shorter and it doesn’t feel as good as the Dutch version (probably because that one is shorter).

I love “delicate as a hand grenade,” too! 🙂

August 21, 2018 at 12:18 pm

Henneke, I love this one: “Because a professional bio is not only about you. It’s about your clients, too.” We have to think more like our own client to help us create a catchy bio that will work and answer “what’s in it for me?” Thanks for the info and the great examples here Henneke. I’ll be playing around with mine in the coming days.

August 21, 2018 at 12:40 pm

“Playing around” is a good phrase for writing a bio!

I can’t remember how long I played around with mine, but it was weeks rather than days (and writing this post makes me wonder whether I have to revisit my Twitter bio again!).

Happy playing, Lisa, and thank you for stopping by.

August 22, 2018 at 10:48 am

Oh yes, I try to update my Twitter bio every month or so. Things change, you know? You are most welcome. Love your mugs by the way as I’ve seen them via Sue-Ann.

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I never saw myself as a writer, but in my early forties, I learned how to write and discovered the joy of writing. Now, I’d like to empower you to find your voice, share your ideas and inspire your audience. Learn how I can help you

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15 personal bio examples that are fun, creative, and get the job done.

Struggling to write about yourself? These personal bio examples show how you can talk about your professional and personal life in a creative, engaging way.

Published on Apr 23, 2024

By Austin Distel

Personal bios can be hard to write. Waxing lyrical about yourself can be uncomfortable, especially in the context of professional achievements and amazing things you’ve done.

But, in a world where we’re often separated from other humans via a screen, your personal bio is one of the easiest ways for a company, colleague, or a potential lead to learn more about you.

‍ If you don’t know what to include or how to get started, we’ve pulled together some personal bio examples that showcase a handful of creative and engaging ways to present yourself on your social media accounts, your personal website, and on any bylined articles you write outside of your site. 

We also provide some cool personal bio generator templates from our AI marketing platform , Jasper. Now you won’t have to scramble around at the last minute to come up with a short bio of yourself when you’re suddenly asked to send one over via email!

The business case for personal bios: why you should write one

You might wonder what the big deal is—it’s just a bio, right? Do people even read them? The answer is actually, yes; they do. Personal bios can be a great way to find out more about the person behind a website or an article. This helps establish a connection and encourages readers to relate to you. 

The thing is, you might not think a lot of people care about where you went to school or what you like to do in your spare time, but you just don’t know who’s reading your professional bio.

It might be a hotshot manager for your dream company who also loves to spend their weekends kayaking the coast. Or it might be the marketer for a well-known brand looking for a writer .

Learning little nuggets of information about you can put you ahead of other candidates they’re researching or other freelancers they’re in talks with. It could be the difference between getting that contract and not. 

What’s the difference between a personal bio and a resume?

Your resume is a list of your professional roles. Your personal bio digs deeper than this and gives readers an insight into things like: 

• Your interests outside of work
• Your backstory
• What gets you up in the morning 
• Your passions and values 
• Your goals 

Understanding this information can help them see if you’d be a good fit, or if you have the personal qualities they’re looking for in someone they want to work with. This information also helps you connect better with like-minded people.

Say, for example, you’re scrolling through Twitter, see a fun Tweet, and click on the person’s bio only to find out they also run 5km every evening and are passionate about addressing climate change. Chances are, you’re going to want to connect with them. 

Elements of a powerful personal bio

Crafting a compelling personal bio is essential for making a strong impression. Your bio should be concise yet impactful, providing a glimpse into who you are and what you stand for. Let's dive into the components that make a personal bio truly powerful:

Relevant personal and professional information

Incorporating both personal and professional details in your bio is key to offering a holistic view of your identity.

Sharing your hobbies, interests, and background not only showcases your unique personality but also makes you more approachable and adds a personal touch to your profile. Personal stories that align with your values and professional repertoire are a good way to connect with your audience.

On the other side of the same coin, showcasing professional details like work experience, skills, and achievements highlights your expertise and establishes your credibility within your field.

Combining personal and professional aspects in a personal bio creates a compelling narrative that offers a comprehensive view of an individual. This powerful blend paints a well-rounded picture, demonstrating both the human side and the professional prowess of the individual, making the bio more memorable and impactful.

Achievements and credentials

Showcasing your achievements and credentials is fundamental to establishing your credibility upon first impression. It is important to recognize that individuals reviewing your profile may only briefly examine your bio, so highlighting these achievements and credentials is imperative. Make sure to present a comprehensive snapshot of your professional capabilities and successes, reinforcing your position as a distinguished expert in your field.

Call to Action (CTA)

Including a call to action in your personal bio, like encouraging visitors to click a website link, is crucial for engaging your audience and motivating them to act.

By offering a clear and concise direction on the desired next steps for the reader, you effectively guide them towards delving deeper into learning about you, your work, or the services you provide. This will not only enhance the user experience by removing any ambiguity but also strategically guides interest towards areas you wish to highlight, like the next step in the sales funnel or a high converting blog article.

Well-crafted calls to action can boost your website traffic, foster engagement, and play a pivotal role in meeting your objectives, whether that’s attracting more clients, expanding your follower base, or unlocking new opportunities.

It's a powerful way to make your bio bring you closer to achieving your goals and shouldn’t be missed.

How to write a personal bio

Before we share some creative bio examples, let’s take a look at what your personal biography should include. Bear in mind that this will vary depending on what platform you’re posting it on (platforms like Twitter and Instagram have a limited word count).

However, you can have a “master” bio that you pull snippets from for other platforms. Not only does this keep your messaging cohesive across platforms, but it makes it easy to get a bio together at short notice because you already have all the information you need written down. 

How to write an Instagram bio

To write an Instagram bio that drives traffic to your website, you need to write a concise ( 150 characters or less ) and engaging description of yourself or your brand. 

Begin with an engaging hook or tagline to capture the reader's interest immediately.

Then, emphasize what distinguishes you from competitors, showcasing your unique selling propositions. Entice visitors with a compelling call-to-action to explore further by clicking the link in your bio to access your website. Use emojis or symbols to add flair and break up the text, injecting personality into your message.

How to write an X (Twitter) bio

It's essential to keep your X (Twitter) bio concise and impactful. Twitter bios have a character limit of 160 characters , so you’ll need to keep things short and sweet.

It's important to remember to periodically refresh your bio, making sure it accurately reflects any updates or changes in your business operations. This practice ensures that your bio remains current and relevant, communicating your business's evolution and current focus to your audience. Keeping your bio updated is a simple yet effective way to engage and inform your followers about the latest developments in your business.

Using platforms like Jasper helps you create a bio that grabs attention, boosts engagement, and drives traffic to your business. This strategy is key to building a strong online presence and fostering your business's growth and success.

What to include in a bio on your site

For this ‌exercise, let’s focus on what you need to include in a personal bio on your website. This will be your “master” bio as it’ll probably be the longest and go into the most detail.

People who are poking around on your website have already shown an interest in who you are and what you do, so they’re more likely to stick around and read a longer bio. Here’s what you should include:

• Your name: obviously, you need to state your name. Most people choose to include their full name, including surname, at the start of their bio. However, if your website URL or personal brand is already your full name, you can shorten it to just your first name. 
• Your position: your current position and the tasks you’re responsible for should get a mention. This is particularly important if you’re looking for jobs or are writing a bio for a networking platform like LinkedIn. 
• Your work experience: mention any key roles you’ve held in the past and how you got to where you are today. Stories like this show readers your journey and give an insight into your specialisms and professional skills. 
• What you do and who you do it for: kind of like stating your “niche”, talking about what exactly it is you do (like graphic design or paid ad campaigns), and who you do it for (like finance companies or busy e-commerce owners) can help readers identify if you’re a good fit. 
• What you do outside of work: personal bios should be more personal than a resume or professional bio and should therefore include personal details about what you like to do when you’re not working. Do you frequent pop-up restaurants? Attend dog shows with your puppy? Rock climb in the mountains? 
• Professional accomplishments: talk about what you’ve achieved both at work and outside of work. This gives readers an idea about the things you value and what you might be able to achieve for them (it’s also a great talking point for people who want to reach out). 
• Personal story: share a personal, creative story to add a splash of personality. It can be anything from the disastrous first birthday party you held for your daughter to the time you were taken in by a local family after getting lost in Mongolia. Don't forget to add a custom image !

How often should you refresh your personal bio? 

The life you live now isn’t the same life you were living ten, five, or even two years ago. Your personal bio should reflect your current situation, which means it’ll probably need to be regularly updated.

If you’re not sure about when to give it a refresh, consider:

• When you land a new job 
• When you achieve a big milestone (either at work or in your personal life)
• When your old one doesn’t have the same ring to it 

Schedule time every few months to go back over your personal bio and see if everything is still true and up-to-date. 

Where to display your personal bio 

Your personal bio can be displayed anywhere that you might want people to find out more about you. Often, this will be places like social media, your website, guest post bylines, or your speaker profile, but you can also include it in other marketing materials like flyers for events you’re hosting or the blurb for your latest ebook. 

• Twitter: cramming a personal bio into 160 characters can be hard, but it can also be a great lesson in identifying what information matters the most. Use up as many of the characters as you can, and use them wisely..
• LinkedIn: the summary section of your LinkedIn profile gives you up to 2,000 characters to use. This is the ideal place to share your “master” bio.
• Instagram: at 150 characters, you have just a few words to explain who you are and what you do on Instagram—use them wisely.
• Personal website: people who land on your website are already interested in you—share your “master” bio here in all its glory. 
• Guest posts: bylined articles need a couple of sentences about the author. Make it engaging to encourage readers to click through to your site or remember your name. 
• Speaker profile: if you speak at events or conferences, you’ll need a short but sweet speaker bio that gets attendees interested in your session.

Get inspired: fun personal bio examples 

#1 twitter bio example: sally fox.

Sally includes what she does and who she does it for, as well as a humorous bit of information about her and her contact information—all within Twitter’s 160 character limit.

#2 Twitter bio example: Kash Bhattacharya

Kash showcases his accomplishments as well as a bit of backstory about what he’s doing and how long he’s been doing it.

#3 Twitter bio example: Jay Acunzo

Jay shares his mission in his Twitter bio, as well as what he’s best known for.

#4 Pinterest bio example: Grey & Scout

Liz of Grey & Scout introduces herself and tells her followers what they can expect to see from her—not an easy feat with such a limited word count.

#5 Guest post bio example: Justin Champion

In this guest author byline, Justin uses the third person to introduce who he is and what he does, as well as share his goal with readers.

#6 LinkedIn bio example: Katrina Ortiz

Katrina uses her LinkedIn bio to tell a story. Readers are hooked from the first sentence, but she also gives key insights into her specialties and achievements. 

#7 LinkedIn bio example: Karen Abbate

Karen does things a bit differently with her LinkedIn summary and lists out key things readers might want to know about her and her career. 

#8 LinkedIn bio example: Katie Clancy

Katie starts her LinkedIn bio with an analogy that serves to hook readers from the start. From there, she branches out into how it relates to her profession, as well as her key achievements and career highlights.

#9 Personal website bio example: Dave Harland

Dave Harland’s About Page shares the story of how he got into writing‌. At the end, he highlights his experience and why this story has helped him become a go-to copywriter.

#10 Personal website bio example: Gummi Sig

Gummi Sig begins his bio in the third person before diving into first-person storytelling mode. Perhaps the best part about this personal bio is the call to action at the end that encourages potential leads to get in touch.

#11 Personal website bio example: Leigh Whipday

Leigh Whipday of Toy Fight has a short professional bio on the website. It includes both his professional achievements as well as insights about his life outside of work to attract his target audience.

#12 Personal website bio example: Blake Fili Suarez

Blake includes both a short and a long version of his bio so that readers can choose the best option for them. Both include what he does as well as personal anecdotes about his life and business.

Create your own with the Jasper personal bio template

# 13 Personal website bio example: Kaleigh Moore

Besides a short professional bio that’s more focused on her work, Kaleigh Moore also has a personal bio on her site. It goes into detail about her first e-commerce business and how it led her into writing for the biggest names in e-commerce. It also shares some fun facts and photos of Kaleigh.

#14 Instagram bio example: Carlos Gil

Carlos Gil does a good job of using limited space wisely, mentioning accomplishments, what he does and for who, as well as what he’s working on.  

#15 Instagram bio example: Samantha Anderl

Co-founder of Harlow, Samantha Anderl, uses her personal Instagram bio not only to promote the company but also to give some insight into her personality. As this bio shows, a little humor can go a long way in attracting like-minded people to both you and your business.

Struggling with a blank page? Not sure which words to put down first? Overwhelmed by all the great professional bio examples out there and have now got writer’s paralysis? That's where an AI writing assistant can come in handy. Jasper’s personal bio template, in particular, can save the day. 

Simply plug in a few key pieces of information about yourself (use the list we mentioned above as a starting point) and choose the tone of voice you want to portray . Jasper will then work its AI magic and create a collection of personal bios you can tweak or use as they are. For example, here’s the start of a short bio for Apple’s co-founder, Steve Jobs. 

Jasper can create well-written, engaging bios for anyone in any role, as long as you provide the right info. For instance, besides setting the point of view and tone, we gave Jasper some basic details, including a fictional name, role, and location for a Senior Product Marketing Manager. 

As you can see from the bio options below, Jasper added emotion and personality to the details we gave. And he even added missing details such as past companies and years of experience, which we could easily swap out for factual details when writing a bio for a real person. 

We provided similar details for the bio of a fictional social media marketer, feeding Jasper info on specialties, previous roles, and hobbies. This time in the third person, Jasper added flair to our inputs, as well as some additional details that could be verified or swapped out, such as Luisa being from Columbia. 

Any of Jasper’s outputs could be combined, edited, or expanded in minutes to create a polished bio that showcases your areas of expertise, your professional goals, and your personality. Writing about yourself doesn’t have to be hard!

Also related: the Jasper company bio template

Companies can also make use of Jasper’s bio templates with the company bio template. Again, just input some key facts about your business, and— voila! —Jasper will create a fun and creative bio you can use wherever you like. 

Create your punchy personal bio today

Never again be asked to send over a personal bio “on the fly”. Instead, create a “master” bio that can be chopped up into engaging chunks and shared on your chosen promotional platforms.

Include key information about your job title, achievements, and what exactly it is you do, as well as personal anecdotes, your interests, and how you spend your spare time.

Your personal bio is a chance to connect with potential clients, hiring managers, and like-minded colleagues, so take the time to write one that really presents you as you want to be presented.

‍ Start a free trial to create your Personal Bio using Jasper today.

Meet The Author:

Austin Distel

Austin Distel is the Sr. Director of Marketing at Jasper , your AI marketing co-pilot. When not working, Austin is also an Airbnb superhost in Austin, Texas.

Enjoy this post? Join over 4 million people who are learning to master AI in 2024.

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How to write a speaker bio (with examples)

• Carolyn Manion Kinnie
• December 9, 2023

Table of Contents

Introduction.

Nailing down the first impression is an essential skill for professional speakers. Because of the high supply in the speaking industry, a first impression that sets you apart from the crowd goes a long way toward landing gigs. Often, knowing how to write a speaker bio is an essential part of creating that incredible first impression.

What to include when writing a speaker bio

If you want to make a good first impression with your bio, it needs to include the following:

• Your name and title (obviously)
• Where you work or serve currently (even if that’s your own business!)
• Your expertise or experience (certifications, degrees, etc.)
• Career highlights (awards, recognition, accomplishments)
• A personal touch (family, hobbies, or fun fact)

Talking about yourself might be easy, but that doesn’t mean you know how to write a bio. And if waxing eloquent about your own accomplishments doesn’t come naturally or this is the first time you’ve put together a professional bio, this task starts to look pretty daunting. Like any professional bio, your speaker bio deserves a good deal of thought and intentionality. Your speaker bio should be oriented toward a few particular goals (which we’ll cover shortly). However, it is far more than a paragraph version of your resume or CV. So what is it? How do you write a speaker bio?

The Speaker Lab exists precisely to help speakers like you with questions like these. We’ve created this guide to writing your speaker bio so you can wow clients and audience members alike, whether you have 100 or 500 words to tell your story!

There are several situations that call for speaker bios. These might vary in length, tone, and audience. However, the principles behind a good professional speaker bio remain the same regardless! In this piece we’ll review the contexts in which you’ll use a speaker bio as well as the principles that make them great. Of course, we can’t tell you what makes a speaker bio fundamentally awesome without giving a few examples! In the second section we’ll dig deeper into some example bios from professional speakers who embody the fundamentals. You can use these fundamental principles as a basic bio template for writing your own!

Find Out Exactly How Much You Could Make As a Paid Speaker

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Where to use your speaker bio

If you’ve read, watched, or listened to a lot of our TSL content, you know one of the first things we tell aspiring speakers to do is create a speaker website. While you’re working on that website, you will realize you need to say something about yourself. The “professional bio” you fired off a couple years ago to your class alumni update probably isn’t going to cut it. Time for a speaker bio!

If you have a standalone speaking website, your bio probably belongs on your “about” page. If your website includes a lot of services other than speaking, you could put a more speaking-oriented bio on your “speaking” page. Don’t sweat this decision too much, because you can always update or shift things around in the future! For inspiration, see how the speaking stars whose bios we feature below tackle this one depending on the scope of their business.

The speaker bio on your website should be directed at your clients. Especially at the beginning of your career, the people who pack in the auditorium will probably not be the people hiring you. A good speaker bio can make or break your chances at landing a gig, so don’t feel that any time or effort spent on this step is wasted! It’s often wisest to have a short, punchy, introduction to your bio as well as a longer, more elaborate section. The short section can go on a landing page, home page, or just the top of your “about” page to capture attention. Check out Erick Rheam’s website to see what we mean!

Another place online where your speaker bio might appear? The website of any Speakers Bureaus you work with.Likely the most professional version of your bio, this will be very accomplishments-oriented as it appeals to clients who already have very specific goals. For a great bio example from a bureau website, look no further than Stephen Shapiro’s at AAE speakers .

So your elegantly crafted “about” page just won you a client. What about they ask for a bio to put in the event program?

Can you just copy and paste from your website? If your website bio is really concise, that might work. Oh no. They asked for ~100 words! Don’t feel bad if you have to pare it down a bit. You can still maintain all the principles of a great speaker bio (see the next section) while targeting a new purpose and readership! If someone has to choose between your talk and another scheduled at the same time, that paragraph (hopefully next to a stunning headshot) has to hook them.

Unlike your website bio, the program bio is specifically oriented toward your audience rather than the event planner. The great thing about these event-specific bios is you can change them up for every audience. Certain awards and achievements with which you wooed the interest of the client might not apply to the very niche industry conference you’re speaking too. They are more interested in how you can serve their specific set of needs, so edit accordingly. This bio will probably have the most constrained word limit of all, but you can still integrate all the principles we list below!

Tip: Make this version of your speaker bio sound so good your event planner doesn’t feel tempted to make any unnecessary revisions. You don’t want to be surprised by what you read about yourself!

How to write a great professional speaker bio

Every speaker brings something unique and wonderful to the industry. That means every speaker bio can and should sound completely different. In this world, standing out from the crowd is your biggest advantage!

That isn’t to say there aren’t any important guidelines to follow. If you peruse the professional bios of the most successful, interesting speakers, they all have a few things in common. We’ve distilled those commonalities into five fundamental principles for a great professional speaker bio. Use these as your bio template and you’ll be off to a great start!

• Engage the reader. Whether it’s a client or a conference attendee, they have read a thousand boring speaker bios before. Be interesting! Put a little extra effort into your writing so it actually reads like a story. Creative entrepreneur Beth Inglish nails this one to a T on her website.
• Maintain your personal brand. Who you are onstage should just be an extension of who you are offstage. If that means lots of data, hard-hitting facts, and no-nonsense tone, that is exactly how to write your bio. We say this all the time…if you’re a speaker, you’re a salesperson…for yourself! Your entire business should radiate authenticity, starting with your bio. For example, many tips for professional bio writing recommend only using the third person. Many speakers buck this trend because they emphasize relatability and connection throughout their personal brand. And that’s totally fine.
• Explain who you serve and what value you add. These are the two essential components of any good speaker bio–and really, any professional bio regardless of your industry. These make up the “hook” that grabs the attention of audience and clients alike. If you cut out the rest of your speaker bio, this section (ideally one or two sentences) should be able to stand alone and still sell your services. At the same time, someone who is not a good fit for your speaking services can read the couple lines of your bio and say “ok, they’re cool but they’re not for me,” saving you both valuable time.
• Include your relevant accomplishments. If you won a speaking award, put it on there! If you have helped business leaders double their revenue in a span of six months, let them know! While you shouldn’t over-inflate reality, this isn’t the time to be overly humble. However, the word relevant is pretty important here. Don’t mention niche accomplishments that neither relate to your audience or that personable, human appeal. Mike Kim , Joe Hirsch , and Nancy Duarte all have amazing speaker bios that integrate their most relevant personal accomplishments.
• Be human. A bio is probably the first glimpse a client gets at what makes you you. If you have some cute pets, love going to Disneyland with your family, or tend to pick up really outside the box hobbies, include it! Many speakers stick these sorts of “fun facts” toward the end, but feel free to integrate them in creative ways as well.

Using these five principles as a rough bio template, you should be able to craft a pretty great description of how awesome of a speaker you are. If you still need inspiration, keep reading!

Amazing examples of how to write a speaker bio

The great bios we mentioned above as examples deserve a closer look. These speakers and friends of TSL embody just about all of the principles we teach for speaking success, so we encourage you to take a look around their sites to see how they do it!

Beth Inglish

Beth’s opener is enough to interest anyone looking to get “unstuck.” She takes full advantage of her website’s “about” page to regale us with an appropriately longer bio. Like a poem, this example is moving and eloquent, describing her path from artistry to speaking. It pulls at the heartstrings and really makes you think about the relationship between art, creativity, and business. Which is exactly what Beth does as a speaker. Beth’s accomplishments are deftly woven into her story rather than advertised front and center, which is perfectly in keeping with her personal brand as a creative. While her masterpiece would have to be pared down for an event program, it is a stunning example of a human, engaging, and authentic professional bio for a speaker website .

Erick Rheam

Erick’s shorter “about me” section includes only the most essential biographical details along with what he does and whom he serves. Any potential client would read it and immediately know if Erick has potential to fit their needs. The longer bio delivers the “why” for Erick’s speaking services, all while seamlessly weaving his professional accomplishments into his speaking journey. If you scroll down, you’ll see Erick includes his personal running bests. His college running career might seem totally unrelated to the speaking industry, but doesn’t that info make him more interesting?

Stephen Shapiro

Compared to some of the other bio examples, Stephen’s might come across as excessively dry. But context is everything! The client who reaches a speakers bureau website likely has very specific needs and wants to know for sure that Stephen is a good fit. This bio gives a comprehensive understanding of his business experience, speaking qualifications, and former clients. Furthermore, this bio clearly and accurately reflects Stephen’s personal brand. If you check out his personal website , he uses copious business-oriented diagrams and references his bigger clients front-and-center.

Mike Kim’s speaker bio is a great example of using relevant accomplishments to make a good first impression with potential clients. He starts with a pithy sentence that captures the audience he serves and the value he adds. Branding is a competitive industry and Mike knows that his potential clients want to see qualifications. As a result, he emphasizes his impressive media and speaking experience. It’s important to note how Mike utilizes a separate landing page for his speaking business. Mike does a lot more than speak, but doesn’t want his other offerings to distract potential clients. His speaking page is linked in the menu of his personal website and offers a distraction-free, simple design showcasing this very to-the-point bio.

Joe’s bio is a great example of how to capture all the principles we mentioned above with brevity. In just a few short paragraphs, he communicates what he does and why he’s qualified to do it. His opening “hook” is an incredibly short sentence that still comprehensively represents his services and audience. While the bulk of the text comprises relevant accomplishments, he doesn’t leave out a reminder that he’s human and has hobbies and a family.

Nancy Duarte

For another incredible example of a short and impactful bio, take a look at Nancy Duarte. A huge part of Nancy’s personal brand is how she built up a successful communications business that works with big time clients. As a result, her bio absolutely exudes success–in less than 150 words! Like Joe above, she still includes a light-hearted line about her family that instantly appeals to grandparents and dog lovers alike. If you are working on a tight, professional bio to use as your go-to for speaking programs, contracts, and anytime you’re unexpectedly asked to provide one…look to Joe or Nancy for inspiration!

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We hope that our tips and examples of professional speaker bios have empowered you to write a bio worthy of all your amazing accomplishments. Before you go, here’s one final tip. Are you worried about word limits after trying to integrate all five fundamental principles of an awesome speaker bio? Start with a longer “master bio” that you can update and edit for each and every context. That way, if you become dissatisfied with some element of one version, you can always replace it or freshen it up without having to come up with new content.

You’re awesome. Your bio deserves to be awesome too. What are you waiting for? Start writing!

• Last Updated: February 29, 2024

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Introductory Speech

Introductory speech generator.

It would be considered rude if the speaker of the seminar was not introduced properly to the audience. How else would the crowd know on who this gentleman or lady really is and what his or her background is on that particular subject. By giving an introductory speech of the guest speaker tonight, you are allowing the audience know who he or she is as a person and what he or she has accomplished or achieved in his or her life. You may also see speech examples in pdf

These kinds of speeches are like PRs, they only tell the good parts about you and never the negative or bad parts about you. As the person assigned to give the introductory speech about the person, your only job is to provide basic background information about that person, the speaker will take care of the rest. But giving an introductory speech would be useless if you are afraid of public speaking. It is important that you learn to conquer your fears and rise above the challenge at hand. You may also check out introduction speech examples  to provide you with a better idea on how to write these kinds of speeches.

The job of an introduction speech is to:

• introduce your guest speaker
• create a welcoming, attentive ready-and-motivated-to-listen anticipation in the audience

Just like the appetizer, your job is to entice the crowd and keep them happy before feasting on the main course. Try your best to wow the audience in order for them to get hyped when the main speaker arrives. Although you have your speech laid out for you, try to add some humor and wit and maybe some jokes as an impromptu as a way to break the ice. You may also like presentation speech examples & samples

To prepare your introduction speech you’ll need:

1. the guest speaker’s name.

When you get the full name of the guest speaker, try to make sure that it is correct cause there are times that the spelling might be wrong. After getting their name, ask for the correct pronunciation of the name. Who would like to hear their mispronounced name, right? You may also check out motivational speech examples & samples

2. The guest speaker’s biography

Aside from knowing just the guest speaker’s name, you got to introduce who he really is as a person and why he or she is the right person to talk about this certain topic. State his or her credentials and what he or she has achieved. Let the audience know who they are listening to and why he or she matter. You may also see informative speech examples & samples

3. A surprise

As mentioned before, it is best to keep things in a very light manner. Nothing too serious should even be said when it is just an introductory speech. Add some jokes, laugh a bit, add humor and wit. Whatever you think that will manage to get the audience’s attention, go for it. You may also like speech examples in doc

How to organize your material

• Build excitement or interest by piling one piece of information after another.
• Make the name of the speech and the speaker, the climax and end of your speech.

Let’s pretend, for the sake of showing you how it’s done, that we’ve already gathered up all the material we need to introduce a guest speaker. You may also check out appreciation speech examples & samples

Introduction Speech Example

1. let’s put this speech in context to help you make sense of it.

The setting for this introduction speech is a conference for an organization called “ Women in Leadership” . The audience are primarily women drawn together through an interest in leadership roles. At the end of the speech, the speaker will lead the clapping as the guest takes center stage. You may also see award speech examples

2. Now here’s the speech text

She’s been a stalwart member of “Women in Leadership” for the last fifteen years. Over that time she’s served in every office: secretary, treasurer, chairperson, chief fundraiser, education officer, chief executive officer to name a few and in some roles several times over. You may also like welcome speech examples & samples

Her passionate dedication and commitment to promoting public speaking as an important component of empowerment is simply amazing and inspiring. We estimate that she has personally mentored at least 200 new speakers and has set an extraordinary “yes, you can” philosophy for many more. You may also check out valedictorian speech examples & samples

We see her as capable, confident and fluent – never at a loss for words. But what you probably don’t know is that this woman was once weak, shy, stuttered, broken and damaged. 

How she got from awkward tongue tied silence to an eloquent front line spokesperson is the story she will share with us tonight. Ladies, I give you … Katherine Watson!” You may also see persuasive speech examples & samples

3. Say the speech out loud! Use it as a template!

Try saying it out loud to get the flow of it. If you like it, use it as a model for the introduction speech you need to write.

Tips to make your introduction speech successful

1. consider tone and language use.

How are you going to build up audience interest if you do not seem motivated and interested to talk about him or her in the first place? While drafting your speech, try to consider the use of language. Is it going to be the same as every introductory speech or are you going to try and spice it up a bit? Remember that in this case, the audience matters as well. Learn on who you are talking to and how you make use of language to gauge your audience is up to you. You may also like  speech outline examples & samples

2. Check the length of your speech

Pertinent and pithy – short and sweet is what you want. One to two minutes should be enough. Try it out loud with a timer. Remember that you are simply the appetizer and not the main course. This is not your time, but the time for the guest speaker. You may also check out orientation speech examples & samples

3. Resist exaggerating or “puffing up” the speaker’s achievements

Try not to exaggerate the guest speaker’s achievements. Try no to sugarcoat the truth just to make him or her look good. It should simply be enough so that the audience can get a quick background of the speaker.

4. Always check your facts

Avoid stating on anything that the speaker is not. It will end very badly for you if you do so. If there are some sensitive issues that you know about the speaker, make sure that you ask him or her permission first before stating it out loud for the whole world to hear. You may also see inspirational speech examples

5. Remember you are the support act!

Cover only enough in your introduction to make the coming speech eagerly anticipated. Do not stray into telling the audience what the guest speaker’s speech will cover in detail.

6. Rehearse

Practice makes perfect. Try practicing in front of a mirror or in front of your family and friends so that they can judge you as to see how you are doing. Sometimes, just winging it is not enough. You have to work on it to develop the confidence you need to improve on your delivery and tone more. You can also see self-introductory speech examples to analyze on how this whole different speech is being framed and written.

Usain Bolt Introductory Speech Example

Ladies and gentlemen, good morning.

Today, it is both my honor and privilege to be able to introduce you to a role model of the athletic world, a man of distinction – Usain Bolt.

Born on August 21, 1986 in Jamaica, he has distinguished himself as a world class sprinter and he currently holds the Olympic and World Records for the 100 meters in 9.69 seconds and the 200 meters in 19. 30 seconds . Wow. Amazing. I wish I could run that fast. You may also like student council speech examples

What makes his achievements all the more remarkable is the fact that they were all set at the 2008 Summer Olympics. He eventually became the first man to win all three events in one of the categories in the Olympics since Carl Lewis in 1984 and the first man in history to set world records in all three events at a single Olympics. His name and his achievements in sprinting have earned him the media nickname “Lightning Bolt”. You may also check out commencement speech examples

I am sure that you all know a great deal about his public sprinting life, but there is more to him than just running.

What you don’t know about the “Lightning Bolt” is that he enjoys dancing and is often characterized as a laid-back and relaxed character . Did you know that before sprinting, his first interested sport was cricket? He said and I quote: “that if he was not a sprinter, he would be a fast bowler instead.” But then, he wouldn’t be known as the “Lightning Bolt”, but something else. You may also see special occasion speech examples & samples

Please give a warm welcome to none other than Usain “Lightning” Bolt to share with you some words of wisdom on not giving up.

Even if you are simply the person assigned to give the introductory speech, you are still required to give it your all and be the best at what you do so that it will leave a good impression to the audience that you have respect for the work that you do and that you take it seriously. You may also like examples of writing a short speech

Text prompt

• Instructive
• Professional

Create an Introductory Speech for a new community project.

Write an Introductory Speech for an academic seminar series.

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• Research Article
• Open access
• Published: 31 May 2024

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells

• Stephen Hsien-Chi Yuan 1   na1 ,
• Chih-Ching Wu 2 , 3 , 4 , 5 , 6   na1 ,
• Yu-Chih Wang 7 ,
• Xiu-Ya Chan 2 ,
• Hao-Wei Chu 2 , 6 ,
• Youngsen Yang 8 &
• Hao-Ping Liu   ORCID: orcid.org/0000-0001-5915-5128 1 , 9  

Cellular & Molecular Biology Letters volume  29 , Article number:  84 ( 2024 ) Cite this article

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Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis.

To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs.

Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs.

This study elucidates AGR2’s pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Introduction

Comparative oncology, a burgeoning field dedicated to investigating cancer risk and tumor development across various species, aims to advance human and animal health [ 1 , 2 ]. By investigating naturally occurring cancers in pet dogs, we gain valuable insights that enhance our understanding and management of relevant human cancers. Among these, canine mammary tumors (CMTs) stand out as the most prevalent neoplasms, constituting 50–70% of tumors in intact female dogs, with approximately 50% being malignant [ 3 , 4 ].

Malignant CMTs predominantly originate from epithelial tissues and exhibit diverse histological subtypes, including the most common complex carcinoma (11%) and simple carcinoma (11%) [ 5 , 6 ]. Complex carcinoma consists of both malignant glandular epithelial cells and benign myoepithelial cells, while simple carcinoma comprises a single neoplastic cell type, which mirrors molecular aspects of human breast carcinomas and often signifies a worse prognosis [ 7 ]. Molecular categorization, based on expression statuses of estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2), classifies CMT into four subtypes: luminal A (ER + /HER2 − ; 44.8%), luminal B (ER + /HER2 + ; 13.5%), basal (ER − /HER2 − and a basal marker positive; 29.2%), and HER2-overexpressing (ER − /HER2 + ; 8.3%) [ 8 ]. Dogs with luminal B tumors exhibit higher mortality, metastasis, and recurrence rates in the liver or lungs compared to those with luminal A tumors. Additionally, HER2-overexpressing cancers tend to present with high-grade histology and aggressive clinical behaviors [ 8 ].

Long-term studies indicate that the risk of CMT-associated mortality within two years of diagnosis ranges from 20 to 45% [ 9 ]. Notably, exposure to ovarian hormones is a significant risk factor in CMT development [ 10 ]. Certain breeds, such as Beagles and Maltese, demonstrate a propensity for CMT, while these tendencies may vary geographically [ 11 , 12 ]. Despite the prevalence of CMT, the underlying molecular mechanisms driving CMT pathogenesis remain largely uncharacterized.

To unravel novel proteins associated with CMT pathogenesis, we employed quantitative proteomic approaches to conduct a comprehensive analysis of the proteomes in CMT tissues, along with matching hyperplastic and normal mammary gland tissues [ 13 ]. This investigation led to the identification of Anterior gradient 2 (AGR2) as highly expressed in CMT tissues. Subsequent analyses conclusively demonstrated significant overexpression of AGR2 in various histopathological subtypes of CMT tissues, contrasting sharply with its expression levels in normal or hyperplastic mammary gland tissues [ 14 ]. AGR2 is an ortholog to the Xenopus laevis protein XAG-2, which plays an essential role in the development of the forebrain and the mucus-secreting cement gland [ 15 ]. Notably, canine AGR2 shares 94% of its peptide sequence with human AGR2.

AGR2 functions as a protein disulfide isomerase (PDI) primarily located in the endoplasmic reticulum (ER). It mediates the formation of disulfide bonds, catalyzes the cysteine-based redox reactions, and adds to the quality control of proteins [ 16 , 17 ]. AGR2 is indispensable for the production of the intestinal mucin MUC-2 [ 18 ]. Additionally, AGR2 engages with MUC-5 as a primary client and is co-expressed with the acidic mucin in Barrett’s esophagus and esophageal adenocarcinoma tissue [ 19 ]. Furthermore, AGR2 binding to the epidermal growth factor receptor (EGFR) in the ER is crucial for the delivery of EGFR to the plasma membrane [ 20 ].

Dysregulation of PDIs can result in the accumulation of misfolded client proteins in the ER, triggering ER stress and unfolded protein response (UPR), consequently contributing to the development of various diseases [ 21 , 22 , 23 ]. AGR2 dysregulation has been linked to a range of diseases, including inflammatory bowel diseases [ 24 ], neoplasm development, and cancer progression [ 17 , 25 , 26 , 27 ]. Overexpression of AGR2 is commonly observed in various human adenocarcinomas, where it promotes tumor growth and metastasis, correlating with a poor prognosis [ 28 , 29 , 30 ]. Moreover, upregulation of AGR2 expression is seen in chemotherapeutics-resistant cancer cells, further contributing to drug resistance [ 31 , 32 , 33 , 34 ].

Notably, AGR2 encompasses an N-terminal signal sequence and can be secreted from both non-tumor and tumor cells into the extracellular milieu through the conventional secretory pathway [ 24 , 25 , 35 , 36 , 37 , 38 , 39 , 40 ]. Elevated levels of extracellular AGR2 have been detected in sera, plasma, or urine of human cancer patients, including those with breast cancer, prostate cancer, urothelial carcinoma, nasopharyngeal carcinoma, and ovarian cancer [ 32 , 41 , 42 , 43 , 44 , 45 ], showing a positive correlation with metastasis occurrence and poor prognosis. Along these lines, our previous study demonstrated a significant association between serum AGR2 concentration in CMT-afflicted dogs and distant tumor metastasis, as well as worse overall survival [ 14 ], suggesting the potential utility of serum AGR2 as a prognostic indicator for CMT. Moreover, our findings reinforce the suitability of CMT as a complementary model for investigating AGR2 function in human cancer [ 46 , 47 ]. Significantly, the carcinogenic properties of extracellular AGR2 underscore its potential as a therapeutic target [ 46 ]. Monoclonal antibodies specifically targeting the extracellular AGR2 have shown promising results in human cancers [ 37 , 48 , 49 ].

In this study, we discovered that AGR2, in addition to being secreted, can regulate the release of 14-3-3ε (YWHAE) and alpha-actinin 4 (ACTN4) from CMT cells. Subsequently, the released 14-3-3ε and α-actinin 4 play a pivotal role in promoting the paracrine chemotaxis of CMT cells.

14-3-3ε is an isoform of the 14-3-3 family of conserved regulatory adaptor molecules expressed in all eukaryotic cells. By recognizing specific phosphorylation motifs and interacting with target proteins, 14-3-3 proteins participate in a variety of intracellular processes, regulate the subcellular localization of target proteins [ 50 ], and act as a stress-adaptive signaling hub in cancer cells that governs critical processes, including apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility [ 51 , 52 ]. Notably, 14-3-3ε has been reported as an oncogene in various cancer cell lines [ 51 ] and viewed as a target to modulate drug sensitivity [ 53 , 54 , 55 ].

α-actinin 4 belongs to a family of actin-binding proteins and is a non-muscle alpha-actinin that has long been associated with cancer development and metastasis [ 56 , 57 ]. α-actinin 4 is predominantly expressed in the cellular protrusions that stimulate the invasive phenotype in cancer cells, and overexpression of α-actinin 4 has been noticed to parallel the accretion of drug resistance in several cancer cell lines [ 58 ].

Despite lacking a signal sequence required for conventional protein secretion through the ER-to-Golgi route, 14-3-3ε and α-actinin 4 have been identified in the secretomes of various human cancer cell lines [ 59 , 60 ] and extracellular vesicles (EVs) originating from human cancers [ 61 , 62 , 63 , 64 ]. Moreover, 14-3-3ε and α-actinin 4 may be released directly from the cell membranes or liberated from the EVs to serve as ligands for cell-surface proteins. For instance, 14-3-3ε has been found to function as a soluble mediator critical in the communication between subchondral bone and cartilage in osteoarthritis [ 65 ]. Similarly, elevated levels of serum α-actinin 4 are associated with lymph node metastasis and a poorer prognosis in cervical cancer [ 66 ].

While intracellular 14-3-3ε and α-actinin 4 have been implicated in cancer progression, their extracellular pro-oncogenic roles have not yet been elucidated. Herein we aimed to characterize how AGR2 controls the release of 14-3-3ε and α-actinin 4 using CMT cells as a model system. To our knowledge, this is the first investigation into the AGR2-modulated secretome, involving the unconventional secretion of oncogenic proteins 14-3-3ε and α-actinin 4. Understanding the mechanisms underlying the AGR2-controlled release of 14-3-3ε and α-actinin 4 in response to stress conditions will contribute to the refinement of AGR2-targeting therapeutic strategies for cancers.

Materials and methods

Four CMT cell lines (CMT-U27, CMT-U27e, CF41.Mg, and DMGT), along with two human breast cancer cell lines (MDA-MB-231 and MCF7) were utilized in this study. CMT-U27 and CF41.Mg were purchased from American type culture collection (ATCC; CRL-3456™ and CRL-6232TM, respectively). CMT-U27 originated from a simple carcinoma-subtype CMT obtained from a 14-year-old female Poodle, while CF41.Mg was derived from a mixed-type CMT acquired from a 10-year-old female Beagle. DMGT originated from a complex carcinoma-subtype CMT obtained from a six-year-old female crossbreed dog, originally established by Dr. Shih-Chieh Chang’s team at the Department of Veterinary Medicine, National Chung Hsing University. The identities of CMT cell lines were confirmed by short tandem repeat (STR) profiling analysis.

CMT-U27e, a subline derived from CMT-U27, was obtained after mock selection in parallel with puromycin selection of AGR2-knockout and control CMT-U27 clones (see below). It exhibits elevated levels of AGR2 compared to the original cell line. CMT-U27 and its derivatives were maintained in Roswell Park Memorial Institute 1640 medium (RPMI; Gibco, Thermo Fisher Scientific), while CF41.Mg, DMGT, MDA-MB-231, and MCF7 were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Thermo Fisher Scientific) with high glucose. For maintenance, cells were grown in culture media supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific) at 37 °C in an incubator supplied with 5% CO 2 .

Transfection

For ectopic plasmid transfection, cells were seeded in 6-well plates at the following densities: CMT-U27 (4.5 × 10 5 cells/well), CF41.Mg (2 × 10 5 cells/well), DMGT (3 × 10 5 cells/well), MDA-MB-231 (3 × 10 5 cells/well), or MCF7 (3 × 10 5 cells/well). The cells were allowed to grow for 18 to 24 h before transfection. CMT-U27, DMGT, MDA-MB-231, or MCF7 was transfected with 1.5 µg of plasmids or the mock control using 4.5 µL of Lipofectamine™ 2000 (Invitrogen, Thermo Fisher Scientific). CF41.Mg was transfected with 2 µg of plasmids using 4 µL of jetPRIME ® (Polyplus, Illkirch, France) according to the manufacturer’s instruction.

For siRNA transfection, CMT-U27 or CMT-U27e was transfected with 75 nM siRNA oligonucleotide duplexes using 10 µL of Lipofectamine™ RNAiMAX (Invitrogen, Thermo Fisher Scientific). Following media replacement, transfected cells were cultured for the specified time intervals before further experiments.

AGR2-expressing vectors

The expression vector for canine AGR2 tagged with Myc and His was previously constructed in a pcDNA3.1(+)-A-myc.His backbone [ 14 ], denoted pcDNA3.1-myc.His-AGR2 throughout the text. The expression vector for HA-tagged AGR2 was generated by integrating the DNA fragment encoding canine AGR2 into a pcDNA3.1(+)-HA-C vector via the KpnI and XhoI site. To generate the expression vector for human AGR2, the DNA fragment encoding human AGR2 was obtained from human A549 cells using similar methods and then inserted into a pcDNA3.1(+)-A-myc.His vector, referred to as pcDNA3.1-myc.His-hAGR2 in the text.

siRNA oligonucleotide duplexes

Duplex oligonucleotides of siRNA against canine AGR2 (denoted siAGR2) were synthesized by and purchased from GeneDireX Inc. (Taoyuan, Taiwan). Sequences of two siAGR2 duplexes are listed as follows: siAGR2-1: 5ʹ-GGCCAAAGAUAUCACAGUUTT-3ʹ and 5ʹ-AACUGUGAUAUAUCUUUGGCCTT-3ʹ; siAGR2-2: 5ʹ-GACUCAGACCUAUGAAGAATT-3ʹ and 5ʹ-UUCUUCAUAGGUCUGAGUCTT-3ʹ.

siRNA duplexes against canine YWHAE (denoted siYWHAE) or those against canine ACTN4 (denoted siACTN4) were synthesized by and purchased from Eurogentec (Seraing, Liège, Belgium). Sequences of two siYWHAE and siACTN4 duplexes are listed as follows: siYWHAE-1: 5ʹ-GUUGACAGUUGAAGAAAGATT-3ʹ and 5ʹ-UCUUUCUUCAACUGUCAACTT-3ʹ; siYWHAE-2: 5ʹ-CAAGGGAGGAGAAGACAAATT-3ʹ and 5ʹ-UUUGUCUUCUCCUCCCUUG-3ʹ; siACTN4-1: 5ʹ-CCUUCCAAGCCUUCAUUGATT-3’ and 5ʹ-UCAAUGAAGGCUUGGAAGGTT-3ʹ; siACTN4-2: 5ʹ-GUUGACAGUUGAAGAAAGATT-3ʹ and 5ʹ-UCUUUCUUCAACUGUCAACTT-3ʹ.

Generation of AGR2-knockout (KO) CMT cell clones

The CRISPR-Cas9 technique was used to generate AGR2-KO cells in CMT-U27 by transient transfection method. Single guide RNA (sgRNAs) targeting canine AGR2 , i.e., canine AGR2 sgRNA-1 or AGR2 sgRNA-2, was constructed into the pU6-gRNA.Ppuro vector by the National RNAi Core Facility, Academia Sinica Taiwan (Nankang, Taipei, Taiwan). AGR2 sgRNA-1 and AGR2 sgRNA-2 sequences are as follows: 5ʹ-GAGTGTAAGAGAGGGCGACG-3ʹ and 5ʹ-GTTGGCCAGAGTGTAAGAGA-3ʹ, respectively. CMT-U27 cells were transfected with the plasmid expressing AGR2 sgRNA-1, AGR2 sgRNA-2, or a mock control using Lipofectamin™ 2000 as described above. The transfectants were selected with 1 μg/mL puromycin at 24 h post-transfection for four days. Live cells were serially diluted and seeded into a 96-well culture plate, then grown until a single cell clone was obtained. Two AGR2-KO clones, KO-S10 and KO-S4, were generated using AGR2 sgRNA-1 and AGR2 sgRNA-2, respectively. The null expression of AGR2 was verified by immunoblotting.

Collection of conditioned media and cell lysates

Conditioned media (CM) were first centrifuged at 300 ×  g for 10 min to remove detached cells and further centrifuged at 3000 ×  g for 10 min to remove cell debris. Collected CM was used for following experiments or applied to further processes as indicated elsewhere. Cell lysates were harvested in a homogenization buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, pH 8.0, 1% NP-40) supplemented with the protease inhibitor cocktail (VWR Life Science, USA) and phenyl methyl sulfonyl fluoride (PMSF; Sigma-Aldrich, USA). Protein concentrations of the CM and cell lysates were measured using a BCA protein concentration assay kit (Pierce, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Transwell migration assay

Conditioned media (CM) collected from corresponding cells subjected to various treatments, or fresh media supplemented with recombinant proteins, were added to the bottom well as the attractant (600 µL per well). Cells were suspended in 160 µL of serum-free culture media, stained with 40 µL of Hoechst 33342 Staining Dye Solution (500 nM; Abcam, UK), and then placed in a top hanging insert with a pore size of 8 μm (SPL, Korea) for a transwell migration assay. Cells were seeded into the insert as follows: CMT-U27 (9 × 10 4 cells), CMT-U27e (9 × 10 4 cells), CF41.Mg (6 × 10 4 cells), DMGT (6 × 10 4 cells), MDA-MB-231 (1 × 10 5 cells), or MCF7 (1 × 10 5 cells). Following a 16- to 20-h incubation at 37 °C, cells in the inserts were fixed with 10% formalin at room temperature for 30 min and then washed several times with 1 × PBS. Cells that remained inside the insert (non-migrated cells) were removed with a cotton swap. Images of migrated cells were acquired by using the Leica DMI3000 B Inverted Microscope (Leica, Wetzlar, Germany) equipped with a Zyla 5.5 Megapixel sCMOS camera (Andor Technology, Belfas, Ireland) under a 10 × objective. The number of migrated cells was measured with MetaMorph ® NX Software (Molecular Devices, San Jose, CA, USA) and presented as the mean + SD of three independent experiments.

Proteomics analysis of the AGR2-affected CMT secretome by a gel-enhanced liquid chromatography-tandem mass spectrometry (GeLC-MS/MS)

Serum-free CM samples collected from transfected CMT cells were deprived of cells and cell debris and subsequently concentrated and desalted with Vivaspin ® 20 (GE Healthcare) following the manufacturer’s instructions. Concentrated CM proteins (10 µg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with 0.5% Coomassie Brilliant Blue G-250 (AppliChem GmbH, Germany). Individual gel lanes were cut into 15 pieces, each of which was dehydrated in acetonitrile (Mallinckrodt Baker) and dried using SpeedVac. The proteins were reduced with 25 mM NH 4 HCO 3 (Sigma-Aldrich) containing 10 mM dithiothreitol (Biosynth AG, Switzerland) at 60 °C for 30 min and alkylated with 55 mM iodoacetamide (Amersham Biosciences, UK) at room temperature for 30 min. The proteins were then digested with trypsin (20 μg/mL; Thermo Fisher Scientific) overnight at 37 °C.

The extracted peptides were analyzed by the LTQ-Orbitrap Discovery (Thermo Fisher Scientific) as described in the previous study [ 59 ]. Briefly, peptide extracts were reconstituted in HPLC buffer A (0.1% formic acid; Sigma-Aldrich), loaded across a trap column (Zorbax 300SB-C 18 , 0.3 × 5 mm; Agilent Technologies, Taiwan) at a flow rate of 0.2 µL/min in HPLC buffer A, and separated on a resolving 100 mm analytical C18 column (inner diameter, 75 µm) using a 15-µm tip (New Objective, USA). The peptides were eluted with a 60-min gradient of HPLC buffer B at a flow rate of 0.25 µL/min across the analytical column. Data-dependent mode was used to detect intact peptides at a resolution of 30,000 and 10 MS/MS scans for the 10 most abundant precursor ions were used to acquire data.

For database searching, the obtained MS/MS spectra were analyzed using the Mascot algorithm (version 2.2.04; Matrix Science, Boston, MA, USA). The search was conducted against the Canis sequence database of Swiss-Prot (released on March 2021; selected for Canis lupus familiaris, 25,491 entries) from the European Bioinformatics Institute. The fragment ion mass tolerance was set to 0.5 Da and the parent ion mass tolerance was set to 10 ppm, with trypsin as the digestion enzyme. Up to one missed cleavage was allowed, and searches included the parameters for variable oxidation on methionine (+ 15.99 Da) and fixed carbamidomethylation on cysteine (+ 57 Da). A random sequence database was used to estimate false-positive rates for peptide matches.

The label-free MS quantification

To identify the AGR2-affected secretome, the label-free MS quantification was used to compare the abundance of CM proteins derived from AGR2-expressing CMT cells with those derived from the vector-expressing control. Each CM sample was meticulously analyzed in triplicate, represented as Rep1, Rep2, and Rep3. The quantification of protein abundance was accomplished by calculating the ratio of the peptide-spectrum match (PSM) count attributed to a specific protein against the PSM counts of all identified proteins within the separate replicates. The cumulative protein abundance, represented as the normalized PSM ratio, within AGR2-expressing CM triplicates was divided by the corresponding cumulative value within vector-expressing CM triplicates. This ratio underwent a logarithmic transformation (Log2), denoted as Log2 (A/V). Proteins that displayed a Log2 (A/V) exceeding the mean + 1.5 SD were classified as AGR2-increased, while those falling below the mean − 1.5 SD were designated as AGR2-decreased.

Immunoprecipitation

Depletion of Myc-tagged AGR2, 14-3-3ε, and α-actinin 4 in the CM was conducted by immunoprecipitation (IP). For depletion of Myc-tagged AGR2, CM of CMT-U27 transfected with an AGR2-expressing vector was incubated with Myc-Trap ® (ChromoTek, Germany) according to the manufacturer’s protocol. The slurry of Myc-Trap ® beads (25 µL per reaction) was equilibrated with dilution buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA) three times, and the equilibrated beads were incubated with 600 µL of CM and rotated end-over-end at 4 °C for 1 h. For the depletion of 14-3-3ε or α-actinin 4 in the CM, antibodies (3 μg) against 14-3-3ε or α-actinin 4 were first incubated with Dynabeads™ Protein G (40 µL per reaction; Thermo Fisher Scientific) and rotated end-over-end at room temperature for 20 min. Subsequently, Dynabeads™ Protein G-immobilized antibodies were incubated with 600 µL of CM and rotated end-over-end at 4 °C for 16 h. The beads were retained with a magnet, and the remained CM was collected and used for a transwell migration assay. The beads were washed three times with wash buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.05% Triton X-100), and the IP complexes were resolved with 30 µL of 2 × sampling buffer (100 mM Tris–HCl, pH 6.8, 2% β-mercaptoethanol, 4% SDS, 20% glycerol, 0.04% bromophenol blue, 100 mM EDTA) for immunoblotting analysis.

Trichloroacetic acid precipitation of CM proteins

Trichloroacetic acid (TCA; Merck KGaA, Germany) was used to precipitate proteins in the 1–2% FBS-containing CM that was applied to a transwell migration assay. CM was mixed with TCA (20%v/v) and incubated at 4 °C overnight. The mixture was centrifuged at 14,000 rpm at 4 °C for 30 min. Precipitated protein pellets were washed with 200 μL of ice-cold acetone (Merck KGaA, Germany) twice to remove residue of TCA, and then air-dried and resuspended in a sample solution (1% sodium dodecyl sulfate (SDS), 10 mM EDTA, pH 8.0). Resuspended samples were further sonicated at 37 °C for 1 h to thoroughly dissolve the protein pellets.

Immunoblotting

Protein extracts resolved in 1 × sampling buffer (50 mM Tris–HCl, 1% ꞵ-mercaptoethanol, 2% SDS, 10% glycerol, 0.02% bromophenol blue, 50 mM EDTA, pH 6.8) were boiled at 95 °C for 10 min, and then separated by SDS-PAGE with 9% to 15% gradient polyacrylamide gels. Protein samples were transferred to POLYSCREEN ® polyvinylidene difluoride (PVDF) membranes (PerkinElmer Life Science, Inc., Boston, MA, USA), blocked with BlockPRO™ Blocking Buffer (Visual Protein, Taiwan) at room temperature for 1 h, and subsequently incubated with appropriately diluted primary antibodies (listed in Table  1 ) at 4 °C overnight.

The membranes were then incubated with secondary horseradish peroxidase (HRP)-conjugated goat-anti-mouse or goat-anti-rabbit IgG (PerkinElmer, at 1:10,000 dilution) at room temperature for 1 h and washed with 1 × Tris-buffered saline containing 0.05% Tween-20 (TBS-T) between steps. Luminescence signals were developed with Western Lightning ® ECL-Pro (PerkinElmer), and images were acquired using Hansor Luminescence Image System (Hansor Polymer Technology Corp., Taiwan) with TSGel software (version 3.5). Quantification of protein bands was conducted using ImageJ (version 1.50i).

Reagents and recombinant proteins

Tunicamycin (Cat. No. NC1771734) was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Rapamycin (Cat. No. 51031-RAP-25) and Chloroquine (Cat. No. 581005-CLQ) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). 3-Methyladenine (3-MA; Cat. No. HY-19312) was purchased from Med Chem Express (MCE, Monmouth Junction, NJ, USA). Recombinant canine AGR2 (rcAGR2) was prepared as previously described [ 14 ]. Recombinant 14-3-3ε (rc14-3-3ε; Cat. No. PKSH031395) was purchased from Elabscience (Houston, Texas, USA).

Immunofluorescence staining and confocal microscopy

Cells were seeded onto coverslips placed in a 12-well plate and grown to 50–70% confluency, and subsequently cultured in serum-free media supplemented with or without 50 nM rapamycin for 16 h. Cells were fixed with 4% paraformaldehyde containing 2% sucrose in 1 × PBS at room temperature for 20 min and then permeabilized with 0.1% Triton X-100 for 3 min and blocked with BlockPRO™ (Visual protein, Taipei, Taiwan) for 30 min. Cells were incubated with an LC3B antibody (83506S, Cell Signaling Technology, Danvers, MA, USA) at 1:100 dilution, together with a 14-3-3 antibody (Cat. No. 11648-2-AP, Proteintech) at 1:400 dilution or an α-actinin 4 antibody (Cat. No. 19096-1-AP, Proteintech) at 1:300 dilution for 90 min at room temperature, followed by staining with secondary Alexa Fluor 488-conjugated goat-anti-mouse IgG and Alexa Fluor 694-conjugated goat-anti-rabbit IgG (Molecular Probe, Thermo Fisher Scientific), respectively, at 1:200 dilution for 45 min. Nuclei were co-stained with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen) at 0.1 µg/mL in 1 × PBS. Coverslips were washed with 1 × PBS between steps and finally mounted with Fluoro-Gel (Electron Microscopy Science, USA) on slides. Images were acquired using the Zeiss LSM780 confocal laser scanning microscope (Jena, Germany) with a 63 × oil-immersion objective.

Quantification of intracellular puncta numbers and colocalization coefficients

The number of LC3B puncta, α-actinin 4-positive LC3B puncta, or 14-3-3ε-positive LC3B puncta, was quantified using ImageJ. Initially, confocal microscopy-acquired images were filtered with the Difference of Gaussian (DoG) filter to enhance the subcellular puncta structure. To quantify the LC3B puncta per cell, individual cells in each image were selected as the regions of interest (ROIs) and subsequently analyzed using the default “Analyze Particles” plugin to count the number of LC3B puncta within each cell.

For quantifying α-actinin 4-positive LC3B puncta or 14-3-3ε-positive LC3B puncta, the default “Colocalization Threshold” plugin was employed to generate overlay images of LC3B puncta and ACTN puncta, or LC3B puncta and 14-3-3ε puncta, prefiltered with the DoG filter, highlighting the overlapping regions. Subsequently, the same ROIs used for quantifying the LC3B puncta per cell in the previous steps were applied to the overlay images, and the “Analyze Particles” plugin was used to count the number of colocalized puncta within individual cells.

The colocalization coefficient was calculated using ZEN (black edition) software from Carl Zeiss AG in Oberkochen, Baden-Württemberg, Germany. To identify LC3B puncta, α-actinin 4 puncta, or 14-3-3ε puncta, the background subtraction thresholds were established based on the fluorescence intensities of the objects. Subsequently, colocalization coefficients between isolated puncta per cell in the overlay images were determined by measuring the overlapping regions between α-actinin 4 puncta and LC3B puncta or between 14-3-3ε puncta and LC3B puncta.

Isolation of extracellular vesicles

CMT-U27e, Ctrl-S3, KO-S10, and KO-S4 were grown in RPMI supplemented with 2% EVs-depleted FBS for 50 h at 37 °C in a humidified incubator with 5% CO 2 supply. For individual cell clones, conditioned media (CM) were collected from cells grown to 80–90% confluency in three 10-cm culture dishes and centrifuged at 300 ×  g for 10 min and subsequently at 3000 ×  g for 10 min at room temperature to remove detached cells and cell debris, respectively. The resulting CM was subjected to the isolation of extracellular vesicles using both differential ultracentrifugation (dUC) and size exclusion chromatography (SEC). For dUC, CM was first centrifuged at 16,500 ×  g at 4 °C for 30 min using the SW 28 Ti Swinging-Bucket Aluminum Rotor (Beckman Life Sciences, Indianapolis, IN, USA). The large EV pellet was washed with 0.22-µm-filtrated DPBS (Gibco, Thermo Fisher Scientific), followed by second centrifugation at 16,500 ×  g at 4 °C for 1 h to remove non-EV contaminants. The remaining supernatants were further centrifuged at 80,000 ×  g at 4 °C for 2 h, and the small EV pellet was washed with 0.22-µm-filtrated DPBS, followed by a second centrifugation at 80,000 ×  g at 4 °C for 2 h to remove non-EV contaminants. The washed large and small EVs were resuspended in 120 µL of 0.22-µm-filtrated DPBS and stored at − 80 °C before further analysis. The remained EV-depleted supernatants were used for a transwell migration assay or immunoblotting.

For SEC, the qEVoriginal/70 nm Gen 2 Column (Izon Science Ltd., New Zealand) was used to isolate EVs. Cell debris-removed CM was first concentrated with Vivaspin ® 20 (100 kD cutoff; GE Healthcare) and then added to the qEV column. Once the CM sample was filled in the column, 500 µL of 0.22-µm-filtrated and degassed DPBS was added to the top of the column. Flow-through fractions were immediately collected as follows: void buffer fractions 1 to 6 and sample fractions 1 to 14 (500 µL each), by the manufacturer’s instruction. The collected fractions were stored at − 80 °C until further use. For a transwell migration assay, 500 µL of 2% FBS-containing RPMI supplemented with 100 µL of the EV fraction (Fraction 1) or DPBS was placed in the bottom well as the attractant.

Nanoparticles tracking analysis (NTA)

NTA was employed to determine the absolute size distribution and concentration of extracellular vesicles (EVs). The analysis was conducted using the NanoSight NS300 instrument (NanoSight, Minton Park, UK) in conjunction with NanoSight NTA software (version 3.4; NanoSight) for both data acquisition and analysis. Particles were automatically tracked and sized based on their Brownian motion and diffusion coefficient. Filtered PBS served as the control and blank samples. The NTA measurement conditions were standardized as follows: temperature maintained at 24.0 ± 0.5 °C, viscosity at 0.99 ± 0.01 cP, frames per second set at 25, and a measurement time of 60 s. The detection threshold remained consistent across all samples. Each sample underwent five recordings to ensure accuracy and reliability of the results.

Transmission electron microscopy (TEM)

TEM was utilized to investigate the morphology of EVs. Initially, isolated EVs were resuspended in 4% paraformaldehyde (50–100 μL), and 10 μL aliquots were deposited onto Formvar/carbon-coated EM grids. The grids were then covered, and membrane adsorption was carried out for 20 min in a dry environment. Subsequently, the grids (with the membrane side down) were transferred to drops of PBS (100 μL) using clean forceps for washing, followed by retransfer to a 50 μL drop of 1% glutaraldehyde for 5 min. Afterwards, the grid underwent eight washes with distilled water, each lasting 2 min. Following this, contrast staining was achieved by immersing the grid in a 50 μL drop of uranyl acetate solution for 5 min. Finally, the grid was embedded in 50 μL of methyl cellulose-UA for 10 min on ice. Upon removal of the grid using stainless steel loops, excess fluid was blotted, and the grid was air-dried. The prepared grid was then examined under an electron microscope (JEM 1230, JEOL Ltd., Tokyo, Japan) operating at 80 kV.

Specimen collection

Pre-surgical serum samples were collected from 17 dogs afflicted with CMT who underwent mastectomy at the Veterinary Medical Teaching Hospital (VMTH), National Chung Hsing University (NCHU), between 2017 and 2019. Additionally, serum samples were collected from 15 privately owned, age-matched healthy female dogs for comparison purposes. Blood samples were drawn into serum separating tubes (SSTs) and allowed to clot at room temperature for 30 min before being centrifuged at 2500 ×  g , 4 °C, for 15 min. The resulting sera were supplemented with a protease inhibitor cocktail (VWP Life Science, Avantor, Radnor Township, PA, USA), divided into 50 μL aliquots, and stored at − 80 °C until utilization.

Paired samples of CMT tissues and non-involved normal mammary gland tissues were collected from 9 out of the 17 CMT patients who underwent simple or bilateral mastectomy at the VMTH, NCHU. These tissues subsequently utilized for collecting the tissue-immersed PBS, mimicking interstitial fluids. All procedures were conducted in compliance with relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee (IACUC) of NCHU (IACUC Number: 109–002). Diagnosis of CMTs was confirmed through radiography and histopathological examination of surgically excised tissues. Classification, histopathological grade, and clinical stage of CMTs were determined on the basis of the modified WHO-TNM system [ 3 ].

Preparation of the tissue-immersed PBS

Paired samples of CMT tissues and non-involved normal mammary gland tissues (5 × 5 × 5 mm 3 ) were harvested during surgical procedures. Upon weighting, the tissues were promptly processed as previously described [ 67 ]. To minimize blood contamination, the tissues were thoroughly washed with ice-cold PBS and then dissected into 1–3 mm 3 fragments using scalpels. The cut tissues were placed into 1.5-mL microcentrifuge tubes and subjected to further washed by ice-cold PBS until the supernatant was clear. Subsequently, the cut tissues were incubated with 600 μL of PBS for 1 h in a humidified incubator at 37 °C containing 5% CO 2 . Following centrifuging at 8000 ×  g for 15 min at 4 °C, the resulting supernatants were promptly treated with a protease inhibitor mixture (2 μL/mL; VWP Life Science) and stored at − 80 °C for subsequent analysis.

Statistical analysis

The statistical analysis was conducted by using GraphPad Prism V8.4 software (GraphPad Inc., San Diego, CA, USA). The two-tailed unpaired t-test was applied to evaluate experimental differences between groups, and the Mann–Whitney U test was utilized to assess differences between clinical samples. p  < 0.05 was considered statistically significant.

Enhanced AGR2 expression promotes CMT cell chemotaxis through modulating extracellular milieu

To investigate the impact of enhanced AGR2 expression on the extracellular milieu of CMT cells, we conducted immunoblotting to assess AGR2 expression status in CMT cell lines: CMT-U27, CMT-U27e, CF41.Mg, and DMGT. CMT-U27 and CMT-U27e displayed an epithelial morphology, characterized by E-cadherin positivity and Vimentin negativity, and expressed the human epidermal growth factor receptor 2 (HER-2) but not the estrogen receptor α (ER-α), as depicted in Fig.  1 A. In contrast, CF41.Mg and DMGT exhibited a mesenchymal phenotype, lacking E-cadherin and expressing Vimentin, and expressed ER-α but little to no HER-2. Endogenous AGR2 was detected in CMT-U27 and found to be expressed at elevated levels in the subline CMT-U27e, whereas AGR2 expression was barely detectable in CF41.Mg and DMGT.

Ectopic expression of AGR2 modulated extracellular milieu of CMT cells, promoting CMT cell chemotaxis. A Characterization of CMT cell lines. Expression of AGR2, E-cadherin, vimentin, HER-2, or ER-⍺ in individual cell lines was analyzed by immunoblotting with specific antibodies. CMT-U27 ( B ) and CF41.Mg ( C ) were transfected with pcDNA3.1-myc.His-AGR2 or the mock vector and grown in 2% FBS-containing RPMI and DMEM, respectively, for 24 h. Whole-cell lysates (WCL) of the transfectants were analyzed by immunoblotting to confirm the expression of Myc-tagged AGR2. Conditioned media (CM) of the transfectants were collected and placed in the bottom well for a transwell migration assay, in which the responding cells were seeded in the top insert and stained with Hoechst during a 16-h incubation. Cells in the insert were fixed for image acquisition using an epifluorescence microscope with a 10 × objective. D , F The number of migrated cells was counted and presented as the mean + SD of three independent experiments. E, G Representative images of migrated cells per field were shown. H Myc-Trap-based precipitation conducted the depletion of AGR2 in the CM of AGR2-expressing CMT-U27. Expression of AGR2 in WCL or CM and Myc-Trap-precipitated AGR2 were verified by immunoblotting. I As described above, the AGR2-depleted CM (denoted deAGR2) or the untreated control was placed in the bottom well for a transwell migration assay. J, K Fresh 2% FBS-containing media supplemented with or without rcAGR2 (800 ng/mL) were placed in the bottom well for a transwell migration assay. For C, F, I–K , statistical significance was determined by a two-tailed unpaired t -test. * p  < 0.05; ** p  < 0.01; **** p  < 0.0001

Subsequently, we transfected CMT-U27, CF41.Mg, and DMGT with AGR2-expressing plasmids to enhance AGR2 expression. Confirmation of ectopic Myc-tagged AGR2 expression in the transfectants was achieved through immunoblotting (Fig.  1 B, C , and Fig. S1A, respectively). To evaluate the functional consequences of altered AGR2 expression, we collected spent conditioned media (CM) of the transfectants and employed a transwell migration assay. Figure  1 D and E illustrate that CM from AGR2-expressing CMT-U27 significantly enhanced the chemotaxis of CMT cells compared with the CM from vector-expressing or non-transfected control cells. Likewise, CM from AGR2-expressing CF41.Mg and DMGT also exhibited chemotaxis-promoting activity (Fig.  1 F, G , and supplementary Fig. S1B, respectively).

To verify the impact of AGR2 on the extracellular milieu of human breast cancer cells, we transfected both invasive MDA-MB-231 and non-invasive MCF7 cells with an expression vector encoding human AGR2. Subsequently, we evaluated the chemotaxis-promoting effect of the resulting CM. MDA-MB-231 expressed undetectable levels of endogenous AGR2, while MCF7 exhibited high levels of endogenous AGR2 expression. The expression of ectopic AGR2 in both cell lines was confirmed by immunoblotting, as shown in Fig. S1C and E, respectively. Furthermore, CM collected from both MDA-MB-231 and MCF7 cells expressing ectopic AGR2 demonstrated chemotaxis-promoting activity compared with CM collected from the vector-expressing or blank control (supplementary Fig. S1D and F, respectively). These findings suggest that the phenomena observed in CMT cells also occur in human breast cancer cells.

To ascertain whether the chemotaxis effect could be attributed to secreted AGR2, we conducted Myc-Trap-based immunoprecipitation (IP) to deplete extracellular AGR2 from the CM of AGR2-expressing CMT-U27. Immunoblotting analysis confirmed the presence of AGR2 in CM trapped by Myc-Trap (Fig.  1 H). We then applied this AGR2-depleted CM (deAGR2) to a transwell migration assay. As depicted in Fig.  1 I, AGR2-depleted CM still retained the ability to attract CMT cells, albeit to a lesser extent than the complete CM. Moreover, the addition of recombinant AGR2 (rcAGR2) to fresh CM showed no chemotaxis effect (Fig.  1 J and K ). These findings suggest that enhanced AGR2 expression modulates components in the extracellular milieu, contributing to CMT cell chemotaxis.

Proteomics analysis unveils the AGR2-affected secretome in CMT cells

To decipher the secretome influenced by AGR2 and its association with chemotaxis, we utilized gel-enhanced liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) to analyze the CM from AGR2-expressing CMT-U27 and CF41.Mg cells, which were effectively transfected, in comparison to their vector-expressing controls (Fig.  2 ). In total, 798 proteins in CMT-U27 and 788 proteins in CF41.Mg were successfully identified and quantified (supplementary Table S1 and  Table S2, respectively). Among these proteins, 51 and 40 CM proteins were identified as AGR2-increased, while 28 and 50 CM proteins were designated as AGR2-decreased in CMT-U27 and CF41.Mg, respectively (Fig.  3 A–C). Intriguingly, seven CM proteins exhibited increased abundance in both CMT-U27 and CF41.Mg cells (Fig.  3 A and C ), including AGR2, 14-3-3ε (gene: YWHAE), and α-actinin-4 (gene: ACTN4), all of which demonstrated prominent increases in magnitude (Fig.  3 A). Furthermore, translin (TSN) was identified as common AGR2-decreased in both CMT-U27 and CF41.Mg (Fig.  3 B and C ).

Identification of AGR2-affected secretome of CMT cells. Schematic diagram for identification of AGR2-affected secretome. Serum-free conditioned media (CM) were prepared from CMT cells (CMT-U27 or CF41.Mg) which had been transfected with pcDNA3.1-myc.His-AGR2 or the mock vector and were grown for another 24 h to 90% confluency. The CM samples were concentrated and resolved by SDS-PAGE and subsequently stained with 0.5% Coomassie Brilliant Blue G-250. Individual protein lanes were cut into gel slices and applied to in-gel digestion, and the resulting peptides were analyzed using a GeLC-MS/MS-based proteomics pipeline. The abundance of identified proteins was determined by label-free MS quantification

Differentially present proteins in the CM of AGR2-expressing CMT cells. A, B Heatmap of the proteins identified as AGR2-increased ( A ) and AGR2-decreased ( B ) in the CM of CMT-U27 or CF41.Mg. Each CM sample was triply analyzed (Rep1, Rep2, Rep3). Protein abundance was determined by normalizing PSMs of specific proteins to total PSMs in replicates. Cumulative normalized PSM ratios of AGR2-expressing CM were divided by vector-expressing CM. This ratio underwent logarithmic transformation (Log2) as Log2 (A/V). Proteins with Log2 (A/V) above mean + 1.5 SD were classified as AGR2-increased; below mean-1.5 SD were designated as AGR2-decreased. AGR2-increased and AGR2-decreased proteins which are in common in both CMT-U27 CM and CF41.Mg CM are highlighted in red ( A ) and blue ( B ), respectively, and are shown in the overlap of Venn diagrams ( C ). D, E Verification of increased 14-3-3ε and α-actinin 4 levels in the CM of AGR2-expressing CMT cells. CMT-U27 ( D ) or CF41.Mg ( E ) cells transfected with the AGR2-expressing or the mock vector were grown in serum-free CM for 24 h, and the CM samples were collected and concentrated. The concentrated CM and whole-cell lysate (WCL) were analyzed by immunoblotting with indicated specific antibodies. Levels of 14-3-3ε and α-actinin 4 in the AGR2-expressing group were presented as fold changes to that in the vector group. Calnexin was used as a negative control in the CM

AGR2 expression augments the release of 14-3-3ε and α-actinin-4 in the CM

Given that 14-3-3ε and α-actinin 4 were among the top-ranked CM proteins increased by AGR2 and were shared by both CMT cell lines, we conducted experiments to validate whether AGR2 expression led to elevated levels of 14-3-3ε and α-actinin 4 in the CM of CMT cells. As depicted in Fig.  3 D and E , ectopic expression of AGR2 in CMT-U27 and CF41.Mg, respectively, resulted in increased release of 14-3-3ε and α-actinin 4 into the CM. Notably, ectopic expression of AGR2 had minimal impact on the protein expression levels of 14-3-3ε and α-actinin 4 in cell lysates. Furthermore, we confirmed that ectopic expression of AGR2 in MCF7 and MDA-MB-231 also enhanced the release of 14-3-3ε and α-actinin 4, as shown in Fig. S2A and B, respectively.

In contrast, ectopic AGR2 expression did not induce the release of an ER protein, Calnexin, in the same experimental context (Fig.  3 D and E ), implying that the observed events were not indicative of a general ER disorganization or rupture caused by ectopic AGR2 expression.

Genetic depletion of AGR2 leads to the diminished release of 14-3-3ε and α-actinin 4 in the CM, alongside impaired chemotaxis activity

We proceeded to investigate whether depletion of AGR2 would impair the release of 14-3-3ε and α-actinin 4 into the CM of CMT cells and influence the chemotaxis activity of the CM. Initially, we employed RNA interference (RNAi) to silence AGR2 expression in CMT-U27e by transfecting with siRNA duplexes targeting the canine AGR2-encoding gene. Immunoblotting analysis confirmed the efficacy of AGR2 silencing, with an efficiency exceeding 75% (Fig.  4 A and B ). Importantly, AGR2 knockdown significantly reduced the extracellular levels of 14-3-3ε and α-actinin 4 (Fig.  4 C and D ), while their expression levels remained unaffected (Fig.  4 A). Subsequently, the transwell migration assay revealed that the CM from AGR2-depleted CMT-U27e exhibited diminished chemotaxis activity compared with the control (Fig.  4 E).

Genetic depletion of AGR2 impaired 14-3-3ε and α-actinin 4 release in the CM of CMT cells. A CMT-U27e transfected with AGR2 -targeting siRNA (siAGR2 1 or 2) or negative control siRNA (siNC) was grown in 1% FBS-containing RPMI for 30 h. WCL and CM samples were collected and subjected to immunoblotting analysis. Protein bands were quantified and normalized to that of GAPDH, which was used as an internal control. AGR2 expression levels in siAGR2 transfectants were presented as a ratio to that in siNC transfectants. Data were presented as the mean + SD of three independent experiments, shown in B . C CM samples collected from ( A ) were TCA-precipitated, and 14-3-3ε and α-actinin 4 levels were analyzed by immunoblotting and quantified as described above. Data are presented as the mean + SD of three independent experiments, shown in D . E CM collected from ( A ) was applied to a transwell migration. Results are presented as the mean + SD of three independent experiments. F, G CMT-U27e, a control cell clone Ctrl-S3, and two AGR2-KO clones, KO-S4 and KO-S10, were grown in 1% FBS-containing RPMI for 24 h. WCL and CM samples were collected and analyzed by immunoblotting, as shown in F and G , respectively. 14-3-3ε and α-actinin 4 levels in individual CM samples are presented as a ratio to that in CMT-U27e, as shown in H . I CM collected from individual cell clones was applied to a transwell migration assay. Results are presented as the mean + SD of three independent experiments * p  < 0.05; ** p  < 0.01; *** p  < 0.001; **** p  < 0.0001 (two-tailed unpaired t -test)

To further evaluate the impact of AGR2 on the release of 14-3-3ε and α-actinin 4, we generated AGR2-knockout (KO) CMT-U27 cell clones using a CRISPR/Cas9-based methodology. Immunoblotting analysis confirmed the complete absence of AGR2 expression in selected AGR2-KO clones, namely KO-S10 and KO-S4 (Fig.  4 F). Significantly, the levels of 14-3-3ε and α-actinin 4 in the CM of AGR2-KO cells were markedly reduced compared with those in the control CMT-U27e or Ctrl-S3 (Fig.  4 G and H ). Furthermore, the CM from AGR2-KO cells displayed substantially diminished chemotaxis activity in comparison to the control CM (Fig.  4 I). These results collectively confirm that AGR2 effectively regulates the release of 14-3-3ε and α-actinin 4 into the CM, which in turn influences CMT cell chemotaxis.

Silencing the expression of 14-3-3ε or α-actinin 4 impairs the chemotaxis activity of AGR2-modulated CM

To ascertain whether reducing the release of 14-3-3ε and α-actinin 4 would diminish the chemotaxis activity of CM, we employed siRNA-mediated RNAi to silence the expression of 14-3-3ε or α-actinin 4 in CMT-U27e. Immunoblotting analysis confirmed the reduced expression of 14-3-3ε and α-actinin 4 in the corresponding siRNA transfectants (Fig.  5 A and B ), accompanied by the reduced levels of 14-3-3ε and α-actinin 4 in the CM (Fig.  5 C and D ). Subsequently, the transwell migration assay revealed that the reduced levels of CM 14-3-3ε or α-actinin 4 resulted in the impaired chemotaxis activity of CM (Fig.  5 E).

Knockdown of 14-3-3ε or α-actinin 4 diminished the chemotaxis conferred by AGR2-modulated CM. A , B CMT-U27e transfected with 14-3-3ε-targeting siRNA, siYWHAE ( A ), α-actinin 4-targeting siRNA, siACTN4 ( B ), or negative control siRNA (siNC) were grown in 1% FBS-containing RPMI for 30 h. WCL samples were harvested and analyzed by immunoblotting. 14-3-3ε and α-actinin 4 levels were normalized to β-tubulin levels in individual samples, and the resulting values in siYWHAE- or siACTN4-transfectants were presented as a ratio to that in siNC. C , D 14-3-3ε and α-actinin 4 levels in the CM samples collected respectively from ( A ) and ( B ) were analyzed by immunoblotting and processed as above. E CM samples collected from ( B, D ) were applied to a transwell migration assay. F, G, I, J CMT-U27e cells were first transfected with the indicated siRNA (75 nM) and subsequently transfected with pcDNA3.1-myc.His-AGR2 or the mock control 6 h later. After 8 h incubation, the culture media were replaced with 1% FBS-containing RPMI, and cells were grown for another 20 h until WCL and CM were collected. Levels of indicated proteins in individual WCL ( F, I ) and CM ( G, J ) were analyzed and quantified as described above. H, K CM samples collected from ( I, J ) were applied to a transwell migration assay. All quantitation data shown are the mean + SD of three independent experiments. * p  < 0.05; ** p  < 0.01; *** p  < 0.001; **** p  < 0.0001 (two-tailed unpaired t -test)

To further elucidate if 14-3-3ε and α-actinin 4 mediate the chemotaxis activity of AGR2-modulated CM, we silenced the expression of 14-3-3ε or α-actinin 4 in CMT-U27 which co-expressed ectopic AGR2. Immunoblotting analysis verified the expression of Myc-tagged AGR2 and the attenuated expression of 14-3-3ε and α-actinin 4 in the corresponding transfectants, as shown in Fig.  5 F and I . The levels of 14-3-3ε and α-actinin 4 were also diminished in the CM of AGR2-expressing CMT-U27 co-transfected with specific siRNAs, as compared with the control (Fig.  5 G and J ). Furthermore, the diminished levels of 14-3-3ε and α-actinin 4 in the CM compromised the chemotaxis activity of AGR2-modulated CM (Fig.  5 H and K ). The cumulative findings demonstrate that AGR2 effectively regulates the release of 14-3-3ε and α-actinin 4, thereby enhancing CMT cell chemotaxis.

AGR2 facilitates the release of 14-3-3ε and α-actinin 4, primarily in non-vesicular form and minorly in extracellular vesicles

Since 14-3-3ε and α-actinin 4 lack a signal peptide required for the conventional ER-to-Golgi secretion, we hypothesized that their release likely occurs through unconventional protein secretion (UPS), such as the extracellular vesicle (EV)-mediated secretion [ 68 , 69 ]. To investigate whether AGR2 regulates the release of 14-3-3ε and α-actinin 4 via EVs, we initially employed differential ultracentrifugation (dUC; supplementary Fig. S3A) to isolate large EVs (lEVs) and small EVs (sEVs) derived from CMT-U27e, Ctrl-S3, KO-S10 or KO-S4. We subjected lEVs and sEVs derived from Ctrl-S3 cells to nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) to confirm the efficiency of EV isolation. NTA results showed that the mean diameters of lEVs and sEVs were 141.7 ± 2.2 nm and 112.3 ± 1.9 nm, respectively (Fig. S3B). TEM images depicted the morphology of the isolated lEV and sEV (Fig. S3C).

Furthermore, immunoblotting results indicated that the levels of 14-3-3ε and α-actinin 4 in the CM were not significantly affected by EV depletion across all the cell lines (Fig. S3D). However, a fraction of 14-3-3ε and α-actinin 4 was detected in lEVs (mainly) and sEVs (minority) derived from CMT-U27e or Ctrl-S3. Notably, the levels of 14-3-3ε and α-actinin 4 in the EVs were significantly reduced in AGR2-KO cells (supplementary Fig. S3E).

To confirm the impact of AGR2 on the delivery of 14-3-3ε and α-actinin 4 via EVs, we conducted size exclusion chromatography (SEC) to isolate the EVs derived from control or AGR2-KO cells (supplementary Fig. S4A). Immunoblotting analysis revealed that 14-3-3ε and α-actinin 4 were present in the CD9-positive EV fractions, particularly Fraction 1, but mostly were detected in the protein fractions (Fractions 9–14) of CMT-U27e (supplementary Fig. S4B), consistent with the previous dUC results. NTA results showed that the mean diameter of EVs was 115.4 ± 2.7 nm, ranging from 70 to 322 nm (Fig. S4C). The morphology of EVs with varied sizes was confirmed using TEM (supplementary Fig. S4D).

Furthermore, we compared the levels of 14-3-3ε and α-actinin 4 in the EV fractions from control cells with those from AGR2-KO cells. The results confirmed reduced levels of 14-3-3ε and α-actinin 4 in the EV fractions from AGR2-KO cells (supplementary Fig. S4E).

To assess the contribution of EV to the CM-driven chemotaxis, we utilized both the complete CM and EV-depleted CM in a transwell migration assay. The results revealed that EV depletion had minimal effect on the chemotaxis activity of CM in all the cell lines (supplementary Fig. S3F), indicating that 14-3-3ε and α-actinin 4 are primarily released as vesicle-free proteins that enhance chemotaxis.

To evaluate the chemotaxis activity of EVs, we subjected EV fractions (Fraction 1) from CMT-U27e, Ctrl-S3, or AGR2-KO cells to a transwell migration assay. Supplementary Fig. S4F illustrated that the EVs derived from AGR2-KO cells displayed diminished chemotaxis activity compared with EVs from control cells, suggesting that the EV-delivered 14-3-3ε and α-actinin 4 might contribute to this effect.

AGR2 controls the release of 14-3-3ε and α-actinin 4 upon ER stress and autophagy induction

Next, we investigated the role of AGR2 in regulating the release of 14-3-3ε and α-actinin 4 during specific cancer progression-related stress conditions, including the unfolded protein response (UPR)-induced ER stress and autophagy [ 70 , 71 , 72 ]. To accomplish this, we exposed CMT-U27e and AGR2-KO cells (KO-S10 and KO-S4) to 50 nM tunicamycin treatment in media containing 1% FBS and then collected cell lysates and CM samples for immunoblotting analysis. Figure  6 A illustrates the upregulated expression of an ER stress indicator, C/EBP homologous protein (CHOP), in response to tunicamycin-induced ER stress in all the tested cells, while basal CHOP levels in AGR2-KO cells declined. Furthermore, tunicamycin treatment led to an increased release of α-actinin 4 and 14-3-3ε from CMT-U27e, but this effect was impaired in AGR2-KO cells (Fig.  6 B; replicate results seen in supplementary Fig. S5A and S5B).

AGR2 modulated the release of 14-3-3ε and α-actinin 4 upon ER stress and autophagy induction. A , B CMT-U27e or AGR2-KO clones (KO-S10 and KO-S4) were cultured in 1% FBS-containing RMPI and treated with or without 50 nM tunicamycin (denoted Tm) for 14 h. Cell lysate (WCL) samples were collected and subjected to immunoblotting analysis with antibodies specific to indicated proteins. The CM samples were further TCA-precipitated to analyze 14-3-3ε or α-actinin 4 levels with immunoblotting ( B ). C , D CF41.Mg transfected with pcDNA3.1-HA-AGR2 or the mock control were subsequently cultured in 1% FBS-containing DMED for 14 h with or without addition of 50 nM Tm. Levels of the indicated proteins in WCL ( C ) and CM ( D ) were analyzed by immunoblotting. E , F CMT-U27e, KO-S10, or KO-S4 were cultured in serum-free RPMI and treated with 300 nM rapamycin (denoted Rm) or 40 µM chloroquine (denoted CQ) for 16 h. Levels of the indicated proteins in WCL ( E ) and CM ( F ) were analyzed by immunoblotting. G , H CF41.Mg transfected with pcDNA3.1-myc.His-AGR2 or the mock control was cultured in serum-free DMEM supplemented with or without 3-MA (2 mM) for 16 h. Levels of the indicated proteins in WCL ( G ) and CM ( H ) were analyzed by immunoblotting. The present results were representative data from three independent experiments

Conversely, tunicamycin treatment induced CHOP expression in CF41.Mg cells and ectopic AGR2 expression intensified this stress response (Fig.  6 C), accompanied by the enhanced release of 14-3-3ε and α-actinin 4 (Fig.  6 D; replicate results seen in supplementary Fig. S5C and S5D). Similarly, these phenomena were also observed in MDA-MB-231 and DMGT cells (supplementary Fig. S2B).

Considering the involvement of autophagy in unconventional protein secretion [ 71 , 73 ], we explored the role of autophagy in the AGR2-controlled release of 14-3-3ε and α-actinin 4. We cultured CMT-U27e and AGR2-KO cells in serum-free media supplemented with 300 nM rapamycin or 40 mM chloroquine, which inhibits autophagosome-lysosome fusion, to induce autophagy. Immunoblotting analysis revealed that rapamycin treatment led to the inhibition of phosphorylation of the mammalian target of rapamycin (p-mTOR), accompanied by a reduction of AKT phosphorylation and CHOP expression in all the analyzed cells (Fig.  6 E). Additionally, chloroquine treatment resulted in the processing of microtubule-associated protein 1 light-chain 3B (LC3B)-I to LC3B-II in all the treated cells, indicating autophagy activation. Notably, both rapamycin and chloroquine treatments facilitated the release of 14-3-3ε and α-actinin 4 in CMT-U27e cells (Fig.  6 F), and similar effects were observed in DMGT, MDA-MB-231, and MCF7 cells (supplementary Fig. S2C). However, this effect was significantly impaired in both AGR2-KO cells (Fig.  6 F; replicate results seen in supplementary Fig. S6A and S6B).

To confirm the involvement of autophagy in the AGR2-controlled release of 14-3-3ε and α-actinin 4, we ectopically expressed AGR2 in CF41.Mg cells and subsequently treated the cells with 3-MA to inhibit autophagy. In Fig.  6 G, it was evident that ectopic expression of AGR2 led to an increased level of p-mTOR, along with enhanced processing of LC3B-II to LC3B-I, compared to the vector-expressing control. In this scenario, 3-MA treatment resulted in an elevated level of p-mTOR, a decreased level of p-AKT, and a reduced LC3B-II-to-LC3B-I ratio in the AGR2-expressing cells. Furthermore, 3-MA reduced the AGR2-controlled release of 14-3-3ε and α-actinin 4 by 0.79-fold and 0.44-fold, respectively (Fig.  6 H; replicate results seen in supplementary Fig. S6C and S6D). These findings reveal that the AGR2-controlled release of 14-3-3ε and α-actinin 4 is intricately linked to ER stress and autophagic processes.

Knockout of AGR2 results in the accumulation of α-actinin 4 and reduced translocation of 14-3-3ε in the autophagosome

To investigate the involvement of autophagic processes in the AGR2-controlled release of α-actinin 4 and 14-3-3ε, we examined the intracellular localization of α-actinin 4 or 14-3-3ε in conjunction with LC3B in CMT-U27e and two AGR2-KO cells under serum starvation or rapamycin treatment. The results revealed that LC3B-positive puncta (representing autophagosomes) were observed in all analyzed cells under serum starvation (Fig.  7 A and B ). Upon rapamycin treatment, the autophagosome numbers were increased in CMT-U27e cells; however, the autophagosome numbers decreased instead in both KO-S10 and KO-S4 cells (Fig.  7 C). In this context, we examined whether α-actinin 4 or 14-3-3ε was translocated to the autophagosomes and if such events were affected in AGR2-KO cells. We used the percentage of autophagosomes containing α-actinin 4 or 14-3-3ε per cell, along with colocalization coefficients between α-actinin 4 and autophagosomes or between 14-3-3ε and autophagosomes, to evaluate the impact of AGR2 on the translocation of α-actinin 4 or 14-3-3ε to the autophagosomes. Under serum starvation, we observed that the percentage of α-actinin 4-containing autophagosomes was increased in KO-S4 cells compared to CMT-U27e cells (Fig.  7 D). Although, in KO-S10 cells, the increase in the percentage of α-actinin 4-containing autophagosomes did not achieve a statistical significance ( p  = 0.08), the colocalization coefficients were significantly higher than those in CMT-U27e cells (Fig.  7 E). Additionally, rapamycin treatment increased the percentage of α-actinin 4-containing autophagosomes and/or the colocalization coefficients in either CMT-U27e or AGR2-KO cells, indicating that the rapamycin-induced translocation of α-actinin 4 was unaffected. Together, these results suggest that AGR2 knockout may impair the autophagic flux and lead to the accumulation of α-actinin 4 in the autophagosomes instead of secretion.

Depletion of AGR2 resulted in aberrant translocation of 14-3-3ε or α-actinin 4 to the autophagosome. CMT-U27e, KO-S10, and KO-S4 seeded on coverslips were cultured in serum-free RPMI for 16 h, with or without adding 50 nM rapamycin (Rm). The cells were subsequently fixed and subjected to immunofluorescence staining of α-actinin 4 ( A ) or 14-3-3ε ( B ), together with LC3B. The resulting images were acquired using confocal microscopy. Puncta exhibiting LC3B (i.e., autophagosomes), α-actinin 4, or 14-3-3ε were identified using a Difference of Gaussian processing filter and presented in overlay images, denoted Merge (P). Scale bar, 20 µm. Colocalization of α-actinin 4 puncta with LC3B puncta, or 14-3-3ε puncta with LC3B puncta, were shown as overlap regions (yellow) in the enlargement, denoted Overlap (P). The number of LC3B puncta per cell ( C ) and the percentage of the LC3B puncta exhibiting colocalization with α-actinin 4 ( D ) or with 14-3-3ε ( F ) across all the experimental groups were quantified. Colocalization coefficients were measured for the puncta showing α-actinin 4-LC3B colocalization ( E ) and those showing 14-3-3ε-LC3B colocalization ( G ) per cell. The data in panels ( C ) through ( G ) were presented as the mean + SD

Furthermore, when examining the colocalization of 14-3-3ε and autophagosomes, we observed that the percentage of 14-3-3ε-containing autophagosomes per cell was lower than the percentage of α-actinin 4-containing autophagosomes in all analyzed cell lines (Fig.  7 F). Although the percentage of 14-3-3ε-containing autophagosomes per cell was unaffected in all analyzed cell lines, the colocalization coefficients were markedly decreased in both AGR2-KO cells compared to CMT-U27e (Fig.  7 G). Collectively, these data imply that the AGR2 knockout may hinder the translocation of a small portion of 14-3-3ε to the autophagosomes, resulting in a reduced release of 14-3-3ε.

Extracellular 14-3-3ε and α-actinin 4 confer the chemotaxis effect of AGR2-modulated CM

To confirm that the chemotaxis activity of AGR2-modulated CM was associated with 14-3-3ε and α-actinin 4, we ectopically expressed AGR2 in both CF41.Mg and CMT-U27 cells. Subsequently, we conducted immunoprecipitation (IP) to deplete 14-3-3ε or α-actinin 4 from the CM or inhibited 14-3-3ε or α-actinin 4 in the CM by incubating with specific antibodies. Immunoblotting analysis confirmed the ectopic expression of AGR2 in both cell lines (Fig.  8 A). We then subjected the CM from AGR2-expressing cells to IP using specific antibodies against 14-3-3ε or α-actinin 4. As depicted in Fig.  8 B, 14-3-3ε or α-actinin 4 in the CM was successfully immunoprecipitated. We then applied the CM samples, both with and without the IP-based depletions, to a transwell migration assay. It was evident that depletion of 14-3-3ε or α-actinin 4 from the CM of AGR2-expressing CF41.Mg or CMT-U27 significantly diminished the CM-driven chemotaxis (Fig.  8 C and D ).

Extracellular 14-3-3ε and α-actinin 4 conferred the chemotaxis effect of AGR2-modulated CM. A WCL and CM samples of CF41.Mg or CMT-U27 transfected with pcDNA3.1-myc.His-AGR2 or the mock control were collected as described previously. Levels of indicated proteins in individual WCL samples were analyzed by immunoblotting. B CM of AGR-expressing CF41.Mg was deprived of 14-3-3ε or α-actinin 4 by immunoprecipitation (IP). Input CM and IP protein products were analyzed by immunoblotting. C , D CM samples before and after IP were subjected to a transwell migration assay. E CM samples of Ctrl-S3 cells grown at sub-confluency were collected and applied to IP, followed by immunoblotting analysis as described in B . F ,  G CM samples of Ctrl-S3 ( F ) or CMT-U27e ( G ) cells supplied with the indicated antibodies (3 μg each) were placed in the bottom well for a transwell migration assay. H CM of KO-S10 supplied with rcAGR2, rc14-3-3ε, or BSA at the indicated concentration was placed in the bottom well for a transwell migration assay. Data of all transwell migration assays were presented as the mean + SD ( n  = 4), and a two-tailed unpaired t-test determined statistical significance. * p  < 0.05; ** p  < 0.01; *** p  < 0.001; **** p  < 0.0001

We then sought to confirm the contribution of 14-3-3ε and α-actinin 4 in the chemotaxis activity of the CM from Ctrl-S3 by inhibiting these proteins with specific antibodies. Initially, we conducted IP to validate the effectiveness of the antibodies in capturing the target proteins within the CM (Fig.  8 E). Subsequently, we applied the CM from Ctrl-S3, supplemented with or without anti-14-3-3ε or anti-α-actinin 4, to a transwell migration assay. The results confirmed that inhibiting 14-3-3ε or α-actinin 4 led to a reduction in the CM-driven chemotaxis (Fig.  8 F). We further explored whether 14-3-3ε and α-actinin 4 cooperated in the chemotaxis activity. For this purpose, we applied the CM from CMT-U27e to a transwell migration assay, incubating the CM with anti-14-3-3ε, anti-α-actinin 4, or both. As shown in Fig.  8 G, inhibiting either 14-3-3ε or α-actinin 4 resulted in a reduction in the CM-mediated chemotaxis. Moreover, inhibiting both 14-3-3ε and α-actinin 4 amplified the reduction in the chemotaxis activity, indicating their cooperative roles in this event. Of note, a portion of α-actinin 4 was co-immunoprecipitated with 14-3-3ε from the CM (Fig.  8 B and E ), suggesting that these proteins may cooperate via a protein–protein interaction.

Considering the availability of recombinant proteins, we first validated the chemotaxis-driven effect of 14-3-3ε in the CM. We supplemented the CM from AGR2-KO cells (KO-S10) with varying concentrations of recombinant 14-3-3ε proteins (rc14-3-3ε). As shown in Fig.  8 H, the addition of rc14-3-3ε to the CM increased the CM-driven chemotaxis, compared with the blank control or the CM supplemented with bovine serum albumin (BSA). In comparison, the addition of rcAGR2 to the CM also enhanced the chemotaxis activity. Notably, we did not observe a dose-dependent or cooperative effect between the CM supplemented with rc14-3-3ε, rcAGR2, or both.

In conclusion, our findings provide evidence that extracellular 14-3-3ε and α-actinin 4 collaboratively contribute to the chemotaxis activity of AGR2-modulated CM. This phenomenon occurs in parallel with the presence of extracellular AGR2.

Levels of 14-3-3ε and α-actinin 4 increase in CMT tissue-immersed PBS and sera of CMT-afflicted dogs

To assess the clinical relevance of the release of 14-3-3ε and α-actinin 4, we initially obtained paired samples of CMT tissues and non-involved mammary gland tissues from 9 female dogs afflicted with CMT (supplementary Table S3). Tissue-immersed PBS samples were collected to extract the tissue-released proteins for subsequent immunoblotting analysis of 14-3-3ε and α-actinin 4. The results revealed elevated levels of both 14-3-3ε and α-actinin 4 in CMT tissue-immersed PBS compared with the paired non-tumor sample (Fig.  9 A). Overall, CMT tissues released significantly higher levels of 14-3-3ε and α-actinin 4 than non-tumor tissues (Fig.  9 B).

Elevated levels of 14-3-3ε and α-actinin 4 in CMT tissue-immersed PBS and sera of CMT-afflicted dogs. A Paired samples of CMT tissues (labeled C) and non-involved mammary gland tissues (labeled N) were obtained from nine female dogs afflicted with CMT. The tissue-immersed PBS samples were collected after one-hour incubation, and the tissue-released proteins were precipitated with TCA for immunoblotting analysis (10 μg of each sample) of 14-3-3ε and α-actinin 4. The CMT sample of patient #4 was replicated in two blots. Results were quantified and presented as fold changes of CMT over paired non-tumor samples. For patient#3, a fold change of CMT was calculated by comparing to the mean of all non-tumor samples. B Elevated levels of 14-3-3ε and α-actinin 4 were observed in CMT tissue-immersed PBS compared with non-tumor tissue-immersed samples. Data from all samples were normalized by the mean of total non-tumor samples. Statistical analysis was conducted using the Mann–Whitney U test. ** p  < 0.01. C Sera were collected from 17 female dogs afflicted with CMT and 15 age-matched healthy female dogs. Each serum sample (1 μL) was diluted in PBS, mixed with 4 × sampling buffer and subjected to SDS-PAGE with two gels. One gel was stained with Coomassie Brilliant Blue G-250, while the other was used for immunoblotting analysis. To quantify the levels of 14-3-3ε and α-actinin 4 in serum samples, the blot intensity of 14-3-3ε or α-actinin 4 was divided by the intensity of the entire lane of proteins stained with Coomassie Brilliant Blue for each sample. The resulting ratio was then normalized by the mean ratio of all healthy samples to acquire a normalized level for comparison. D Elevated levels of 14-3-3ε and α-actinin 4 were observed in sera from CMT-afflicted dogs compared with those in sera from healthy dogs. Statistical analysis was conducted with the Mann–Whitney U test. ** p  < 0.01

Next, we investigated the presence of 14-3-3ε and α-actinin 4 in canine sera and its correlation with CMT. Immunoblotting analysis was conducted to detect these proteins in sera collected from 17 female CMT-afflicted dogs and 15 age-matched healthy female dogs (Table S3, Additional file 3 ). To quantify the levels of 14-3-3ε and α-actinin 4 in serum samples, the blot intensity of 14-3-3ε or α-actinin 4 was divided by the intensity of the entire lane of proteins stained with Coomassie blue for each sample (Fig.  9 C). The resulting ratio was then normalized by the mean ratio of all healthy samples to acquire a normalized level for comparison. The results confirmed elevated levels of 14-3-3ε and α-actinin 4 in the sera of CMT-afflicted dogs compared with those in the sera of healthy dogs (Fig.  9 D), suggesting clinical relevance of CMT cell-released 14-3-3ε and α-actinin 4.

In this study, we discover a novel role for AGR2 in facilitating the unconventional secretion of 14-3-3ε and α-actinin 4, alongside its conventional secretion. This process modulates a pro-chemotaxis extracellular microenvironment (Fig.  10 ). AGR2 has previously been shown to regulate the folding and secretion of its client proteins, e.g., MUC2 and MUC5, through the classical secretory pathway. Our present study unveils the interconnections between the intracellular function of AGR2 and the regulation of unconventional protein secretion encompassing multiple routes (e.g., EV and secretory autophagy) for externalizing proteins that lack a signal peptide, such as 14-3-3ε and α-actinin 4. Unconventionally secreted 14-3-3ε and α-actinin 4 are functional as paracrine (Fig.  8 ), and their extracellular roles could be distinct from their intracellular functions, thereby augmenting their oncogenic impacts.

Schematic illustration of a proposed model for how AGR2 controls the release of 14-3-3ε and α-actinin 4 to promote chemotaxis of CMT cells. AGR2 functions as a stress sensor, regulating proteostasis by controlling the release of unconventional secretory proteins. Upon serum starvation, tunicamycin-induced ER stress, or rapamycin-induced autophagy, AGR2 expression promotes the release of 14-3-3ε and α-actinin 4 into the extracellular microenvironment, thereby enhancing chemotaxis in CMT cells. This controlled release of 14-3-3ε and α-actinin 4 by AGR2 involves extracellular vesicle (EV)-mediated delivery and secretory autophagy. Depletion of AGR2 leads to a reduced release of 14-3-3ε and α-actinin 4 in response to serum starvation, ER stress, or autophagy induction. Additionally, the absence of AGR2 can result in diminished uptake of 14-3-3ε within the LC3B + autophagosome and impaired export of α-actinin 4 through the LC3B + autophagosome

Moreover, we confirmed the AGR2-modulated unconventional release of 14-3-3ε and α-actinin 4 in both CMT and human breast cancer lines representing diverse molecular subtypes, including CMT-U27 and CMT-U27e (ER − /HER2 + , E-cadherin + /Vimentin − ), CF41.Mg and DMGT (ER + /HER2 − , E-cadherin − /Vimentin + ), MCF7 (ER + /HER2 + , E-cadherin + /Vimentin − ), and MDA-MB-231 (ER − /HER2 − , E-cadherin − /Vimentin + ). These findings suggest the influence of AGR2 on the release of 14-3-3ε and α-actinin 4 may transcend specific cell types, potentially affecting a diverse range of cellular contexts.

The molecular mechanism underlying how AGR2 regulates the release of 14-3-3ε and α-actinin 4 currently remains elusive. Apparently, the release of 14-3-3ε and α-actinin 4 is uncoupled from the conventional secretion of AGR2. AGR2 secretion is inhibited by brefeldin A, which inhibits the ER-Golgi vesicle transport, while the release of 14-3-3ε and α-actinin 4 is enhanced by brefeldin A instead (Fig. S6C). In agreement with the notion that the unconventional protein secretion is often linked to cellular stresses [ 70 , 73 ], the release of 14-3-3ε and α-actinin 4 is responsive to tunicamycin-triggered ER stress (Fig.  6 B, D; Fig. S2B, S2C), serum starvation, and rapamycin-induced autophagy (Fig.  6 F; Fig. S2C) that also cause autophagosomes to form (Fig.  7 A–C). Under these stress conditions, however, depletion of AGR2 results in a significant reduction in the release of 14-3-3ε and α-actinin 4 (Fig.  6 B, F ) and the formation of autophagosomes (Fig.  7 C). In contrast, ectopic expression of AGR2 intensifies this stress-related protein secretion (Fig.  6 D; Fig. S2B), which can be diminished by addition of an autophagy inhibitor, 3-MA (Fig.  6 H). Furthermore, the absence of AGR2 likely hinders the engulfment of 14-3-3ε into the autophagosome (Fig.  7 G) and the release of α-actinin 4 through the autophagosome, leading to reduced 14-3-3ε translocation and increased α-actinin 4 accumulation in these LC3B-labeled compartments, respectively (Fig.  7 D, E ). Collectively, these findings suggest that AGR2 may contribute to maintaining balanced proteostasis by regulating the unconventional protein secretion, partially involving the autophagic flux, i.e., secretory autophagy [ 68 ], as depicted in the proposed model (Fig.  10 ).

Although 14-3-3ε and α-actinin 4 have been identified as EV contents, our study demonstrates that AGR2 controls the release of 14-3-3ε and α-actinin 4 via both EVs and non-vesicular routes (Fig. S3D, S3E, and Fig. S4E), with the latter delivering the majority of extracellular 14-3-3ε and α-actinin 4. As a result, non-vesicular 14-3-3ε and α-actinin 4 mainly confer the pro-chemotaxis effects (Fig. S3F), which are impaired by depleting 14-3-3ε or α-actinin 4 from the CM (Fig.  8 C–G) but are increased by supplementing recombinant 14-3-3ε in the CM (Fig.  8 H). It is speculated that cytosolic 14-3-3ε and α-actinin 4 may associate with a chaperon or a membrane receptor facilitating their translocation into the lumen of an autophagosome. Additionally, 14-3-3ε and α-actinin 4 may also be engulfed within an intra-lumen vesicle (ILV), which subsequently fuses with an autophagosome. The 14-3-3ε or α-actinin 4-containing autophagosome eventually fuse with the plasma membrane to release 14-3-3ε or α-actinin 4 in non-vesicular forms or within the ILVs corresponding to EVs, similar to the well-known unconventional secretion of IL-1β [ 74 ]. On the other hand, we observed that only 20% of autophagosomes per cell encompass 14-3-3ε (Fig.  7 D), compared with the observation that 40% of autophagosomes contain α-actinin 4 (Fig.  7 F). This leads us to hypothesize that 14-3-3ε may be preferentially translocated into the endosome or lysosome, which directly fuses with the plasma membrane to release soluble 14-3-3ε, similar to the unconventional secretion of the fatty acid binding protein 4 (FABP4) [ 75 ]. Rationally, both α-actinin 4 and 14-3-3ε protein sequences possess in silico predicted KFERQ-like motifs, as identified in IL-1β and FABP4 protein sequences, which bind to the chaperon or the receptor required for facilitating their translocation into the relevant compartments. Those motifs include 52 QRKTF 56 , 213 DKLRK 217 , 214 KLRKD 218 , 361 QTKLR 365 , 416 EKFRQ 420 , 753 QILTR 757 in α-actinin 4, and 222 QLLRD 226 in 14-3-3ε, as predicted by using KFERQ finder V0.8 ( https://rshine.einsteinmed.edu/ ). Our study suggests that 14-3-3ε and α-actinin 4 could be unconventionally secreted via chaperon-mediated autophagy, and AGR2 appears to play active roles in multiple steps during this process. Further investigation is warranted due to the clinical significance of AGR2 and unconventionally secreted oncogenic proteins.

This study has certain limitations. Firstly, we observed the effect of AGR2-modulated secretome on chemotaxis using a simple culture system in its current state. To rigorously validate these findings, transitioning to a three-dimensional culture of CMT cells co-incubated with recipient cells or an in vivo model is crucial. Moreover, there is currently a lack of clinical correlations between expression levels of AGR2, extracellular levels of 14-3-3ε and α-actinin 4, and the status of CMT. To assess the clinical relevance, we validated elevated levels of 14-3-3ε and α-actinin 4 in the PBS containing CMT tissue-released proteins as well as in sera from CMT-afflicted dogs, utilizing a small cohort of specimens (Fig.  9 ). These results underscore the importance of further endeavors to analyze the correlation between the expression levels of AGR2, 14-3-3ε and α-actinin 4 in tumor cells, as well as the levels of serum AGR2, 14-3-3ε and α-actinin 4 under stress conditions, using specimens from an expanding number of CMT patients.

Recently, 14-3-3ε and α-actinin 4 have been identified in cancer-derived EVs in sera or plasma of cancer patients [ 61 , 62 ], while their extracellular oncogenic roles await thorough investigation. A related study reveals that human colorectal cancer cells release vesicles containing 14-3-3ξ along with β-catenin, which can be taken up by neighboring cells and enhance their migration ability via activation of the Wnt signaling [ 76 ]. Intracellularly, 14-3-3 proteins are known for their regulation of multiple signaling pathways that govern critical processes in cancer via interacting with various partners. The changes in environmental conditions will result in the loss of homeostatic 14-3-3 interactions and trigger new interactions [ 52 ]. However, whether or how extracellular 14-3-3ε reaches proteins other than its intracellular partners to exert cancer-promoting functions remains unclear. In this study, we demonstrate that 14-3-3ε associates with α-actinin 4 in the CM (Fig.  8 B, E ) to promote cell chemotaxis synergistically (Fig.  8 G). It has been shown that extracellular 14-3-3 proteins bind to aminopeptidase N (APN, CD13) and thereby induce the transcription of matrix metalloproteinase genes [ 63 , 77 ]. Further investigation is needed to elucidate if extracellular 14-3-3ε and α-actinin 4 co-opt to bind to APN or other surface receptors of recipient cells in the microenvironment, and how EV-delivered 14-3-3ε and α-actinin 4 transduce relevant signaling pathways in the recipient cells to modulate the tumor microenvironment.

Conclusions

In summary, this present study provides new insights into the roles of AGR2 in coping with the complex interplay between ER stress, pro-survival unfolded protein response (UPR), autophagy, and unconventional secretion of oncogenic proteins, such as 14-3-3ε and α-actinin 4. Sustained ER stress and autophagy have been considered mechanisms underlying tumor progression, metastasis, and resistance to anticancer agents [ 70 , 78 ]. In this perspective, unconventionally released proteins and EV contents from cancer cells due to stress could be oncogenic and favor a pro-oncogenic tumor microenvironment. Further investigation into the impact of extracellular 14-3-3ε and α-actinin 4 on cancer cell phenotypes and therapy responses, coupled with an analysis of their concentrations in cancer patients, may suggest new avenues in the management and therapy of cancers. Addressing secreted 14-3-3ε and α-actinin 4 in addition to AGR2 in the microenvironment could offer novel insights into cancer treatment strategies.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files. Original immunoblots are provided in Supplementary Material 5. The MS raw data for proteome analysis were deposited on the ProteomeXchange Consortium website ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository [ 79 ], data set identifier: PXD047008.

Abbreviations

Alpha-actinin 4

Anterior gradient 2

C/EBP homologous protein

Conditioned media

Canine mammary tumors

Endoplasmic reticulum

Estrogen receptor

Extracellular vesicle

Human epidermal growth factor receptor 2

Microtubule-associated protein 1 light-chain 3B

Mammalian target of rapamycin

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

Unfolded protein response

Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon

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Acknowledgements

We express our sincere gratitude to Dr. Chih-Ching Wu’s team members for their invaluable contributions to the revision of this manuscript, particularly during the periods when the first author was unable to contribute. Additionally, we extend our appreciation to the Microscopy Center at Chang Gung University (Taoyuan, Taiwan) for their technical assistance.

This work was supported by Grants from the National Science and Technology Council of the Republic of China (MOST 109-2320-B-005-011 and MOST 111-2313-B-005-041 to H-PL; NSTC 112–2320-B-182-044 to C-CW) and Taichung Veterans General Hospital/National Chung Hsing University Joint Research Programs (Grant number: TCVGH-NCHU1117616 and Grant number: TCVGH-NCHU1137614 to H-PL and YY).

Author information

Stephen Hsien-Chi Yuan and Chih-Ching Wu contributed equally to this work and shared the first authorship.

Authors and Affiliations

Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan

Stephen Hsien-Chi Yuan & Hao-Ping Liu

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

Chih-Ching Wu, Xiu-Ya Chan & Hao-Wei Chu

Department of Otolaryngology-Head and Neck Surgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan

Chih-Ching Wu

Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan

Department of Medical Biotechnology and Laboratory Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan

Chih-Ching Wu & Hao-Wei Chu

Graduate Institute of Veterinary Pathology, College of Veterinary Medicine, National Chung Hsing University, Taichung, 40227, Taiwan

Yu-Chih Wang

Department of Oncology, Taichung Veterans General Hospital, Taichung, Taiwan

Youngsen Yang

Biotechnology Center, National Chung Hsing University, Taichung, 40227, Taiwan

Hao-Ping Liu

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Contributions

H-PL conceived, designed, and conducted the experiments; analyzed and curated the data; administrated the project and acquired funding; wrote, reviewed, and revised the manuscript. SHY conducted the cell-based experiments, analyzed data, and drafted the original version of the Materials and Methods and Results sections for Figs.  1 , 2 , 3 . C-CW designed the proteomic analysis and experiments involving canine sera, acquired and analyzed data, drafted the Materials and Methods section, and acquired funding. Y-CW provided consultations and technical support for EV collection. X-YC performed the proteomic analysis and analyzed canine serum samples. H-WC analyzed the colocalization coefficient data. YY acquired funding. All authors have read and approved the final manuscript.

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Correspondence to Hao-Ping Liu .

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Supplementary Information

Supplementary material 1: table s1. cmt-u27_protein id and quantification., supplementary material 2: table s2. cf41.mg_protein id and quantification., supplementary material 3: table s3. signalments of healthy female dogs and dogs with malignant mammary tumors, 11658_2024_601_moesm4_esm.pdf.

Supplementary Material 4: Fig. S1. Ectopic expression of AGR2 modulated extracellular milieu of several cancer cell lines to promote cell chemotaxis. The CMT cell line DMGT (A, B) was transfected with pcDNA3.1-myc.His-AGR2 or the mock vector and grown in 1% FBS-containing DMEM for 24 h. Additionally, the human breast adenocarcinoma cell line MDA-MB-231 (C, D) or MCF7 (E, F) was transfected with an expression vector for human AGR2 under similar condition settings. (A, C, E) Whole-cell lysates (WCL) of the transfectants were analyzed by immunoblotting to confirm the expression of Myc-tagged AGR2. Conditioned media (CM) of the transfectants were collected and placed in the bottom well for a transwell migration assay. Cells in the insert were fixed for image acquisition using an epifluorescence microscope with a 10 × objective. (B, D, F) The number of migrated cells was counted and presented as the mean + SD of three independent experiments. Statistical significance was determined by a two-tailed unpaired t-test. *, p  < 0.05; **, p  < 0.01. Fig. S2. AGR2 modulated the release of 14-3-3ε and α-actinin 4 in several cancer cell lines. (A) MCF7 transfected with pcDNA3.1-myc.His-hAGR2 or the mock control were subsequently cultured in DMEM containing 1% FBS for 24 h. The levels of the indicated proteins in WCL and CM were analyzed by immunoblotting. (B) DMGT or MDA-MB-231 transfected with pcDNA3.1-myc.His-AGR2, pcDNA3.1-myc.His-hAGR2, or the mock control were subsequently cultured in DMEM containing 1% FBS for 14 h with or without addition of 50 nM tunicamycin (Tm) following transfection. The levels of the indicated proteins in WCL and CM were analyzed by immunoblotting using antibodies specific to indicated proteins. (C) DMGT, MDA-MB-231, or MCF7 were cultured in 1% FBS-containing DMEM and treated with 100 nM rapamycin (Rm) or 40 µM chloroquine (CQ) for 20 h. The levels of the indicated proteins in WCL and CM were analyzed by immunoblotting. These results represent data from three independent experiments. Fig. S3. Extracellular vesicles were dispensable for the chemotaxis conferred by AGR2-modulated CM. (A) Schematic diagram for comprehensive isolation of extracellular vesicles (EVs) with varied sizes by differential ultracentrifugation. (B) Nanoparticle tracking analysis (NTA) was performed on the isolated large (l) EVs and small (s) EVs derived from control Ctrl-S3 cells. (C) Transmission electron microscopy (TEM) images depict the isolated lEV and sEV (indicated by arrowheads). (D) The CM samples before and after EV depletion, and the WCL of CMT-U27e, Ctrl-S3, KO-S10, and KO-S4, respectively, were analyzed by immunoblotting with antibodies specific to the indicated proteins. CD9 and CD63 are considered markers for exosomes with smaller sizes, and Annexin A1 is considered a marker for microvesicles with larger sizes. (E) Isolated lEVs and sEVs originating from individual cells were analyzed by immunoblotting with specific antibodies as indicated. (F) CM samples with and without EV depletion (denoted EV-present and EV-depleted CM, respectively) were applied to a transwell migration assay. Data are presented as the mean + SD of three independent experiments. Fig. S4. AGR2 knockout led to a reduction in the EV-delivered 14-3-3ε and α-actinin 4 and the EV-mediated chemotaxis. (A) The schematic diagram for isolation of EV-enriched fractions by the size exclusion chromatography (SEC) using the qEV Isolation Columns. (B) CM from CMT-U27e cells was first concentrated with VivaSpin 20 (> 100 kD) and applied to the SEC. Individual fractions collected throughout the SEC, along with the concentrated CM (> 100 kD) and the flowthrough (< 100 kD) subsequently concentrated for the second time, were analyzed by immunoblotting with antibodies specific to 14-3-3ε, α-actinin 4, CD9, or Albumin. Albumin was used as an indicator for the EV-free protein fractions. (C) Nanoparticle tracking analysis (NTA) was conducted on the isolated EVs derived from CMT-U27e cells in qEV Fraction 1. (D) Transmission electron microscopy (TEM) images illustrate the isolated EVs (indicated by arrowheads) in qEV Fraction 1. (E) The CM fractions of CMT-U27e, Ctrl-S3, KO-S10, or KO-S4 collected during the SEC, together with the corresponding input CM (> 100 kD) and the flowthrough (< 100 kD), were analyzed by immunoblotting. (F) The EV-enriched fractions (Fraction 1) derived from individual cells were applied to a transwell migration assay. Data are presented as the mean + SD of three independent experiments, and the statistical significance was determined by a two-tailed unpaired t-test. *, p  < 0.05; **, p  < 0.01. Fig. S5. Experimental replicates of the immunoblotting analysis which are shown in Fig.  6 (A to D). (A, B) Experimental replicates of the immunoblotting data shown in Fig.  6 A and Fig.  6 B. (C, D) Experimental replicates of the immunoblotting data shown in Fig.  6 C and Fig.  6 D. Fig. S6. Experimental replicates of the immunoblotting analysis which are shown in Fig.  6 (E to H). (A, B) Experimental replicates of the immunoblotting data shown in Fig.  6 E and Fig.  6 F. (C, D) Experimental replicates of the immunoblotting data shown in Fig.  6 G and Fig.  6 H.

Supplementary Material 5: Original immunoblots in this study.

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Yuan, S.HC., Wu, CC., Wang, YC. et al. AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells. Cell Mol Biol Lett 29 , 84 (2024). https://doi.org/10.1186/s11658-024-00601-w

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Received : 25 December 2023

Accepted : 21 May 2024

Published : 31 May 2024

DOI : https://doi.org/10.1186/s11658-024-00601-w

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• Canine mammary tumor (CMT)
• Anterior gradient 2 (AGR2)
• 14-3-3 Epsilon (YWHAE)
• Alpha-actinin 4 (ACTN4)
• Unconventional protein secretion
• Microenvironment

Cellular & Molecular Biology Letters

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