PONE-D-24-41815Mechanisms of Adipocyte Regulation: Insights from HADHB Gene ModulationPLOS ONE

Dear Dr. Huang,

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript entitled “Mechanisms of Adipocyte Regulation: Insights from HADHB Gene Modulation” summarized the Adipocyte proliferation and differentiation are vital for energy metabolism and lipid synthesis, ensuring energy homeostasis. The HADHB gene, associated with fatty acid metabolism, was studied for its role in regulating bovine preadipocyte proliferation and differentiation. Findings revealed that HADHB modulates key genes influencing these processes and alters cell cycle progression, providing insights into adipocyte biology regulation. Generally, the manuscript is well written. This paper has several weaknesses and needs improvement before publication.

This manuscript has major language problems. There are too many for me to modify them all. Authors are strongly encouraged to seek a native English speaker who may assist you modifying the document.

Comments:

1. Please write your full affiliation with your college you only written the university name ?

2. What cell type was utilized for RNA extraction

3. How much RNA was used in PCR? Was it one-step or 2-step PCR?

4. The abstract is not particularly informative and would benefit from more background.

5. Summarize the abstract, focus on the main findings and mention the small conclusion in at the end of abstract

6. In the Introduction focus on the objectives and insert a few new reference and relevant findings

7. In material and method sections, references are missing.

8. Most of the references mentioned are old and I suggest adding recent references, and the manuscript should be edited accordingly.

9. I suggest the cite following paper in introduction part For more information you can read below reference

Investigating the role of KLF6 in the growth of bovine preadipocytes: using transcriptomic analyses to understand beef quality. Journal of Agricultural and Food Chemistry, 72(17), pp.9656-9668.Material and method needs to clarifying and summarizing- some detailed needs

The subtitles in the material and method needs to summarizing Ethical approval and references must be mentioned in M&M

Reviewer #2: The purpose of the manuscript "Mechanisms of Adipocyte Regulation: Insights from HADHB Gene Modulation" was to learn more about the mechanisms behind adipocyte regulation while examining the impact of the HADHB gene on the differentiation and proliferation of bovine preadipocytes. The authors performed both knock down and overexpression experiments. Based on the results, it appears that the HADHB gene regulates the expression of adipogenic marker genes CEBPα and PPARγ, which may play a role in adipocyte differentiation. Additionally, the transcriptome study revealed that the transcriptional profile of preadipocytes was considerably altered by HADHB gene overexpression. The HADHB gene, according to the authors, controls PCNA and CDK2 expression, which promotes preadipocyte maturation and proliferation. In addition, HADHB controls the production of CEBPα and PPARγ, which helps to control preadipocyte development and the creation of lipid droplets. This manuscript is very well written. The methods are described in detail and the conclusions are based on the results. Figures are well prepared and well labelled. I congratulate the authors for performing a thorough study to decipher the role of HDAHB in adipocyte regulation.

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PONE-D-24-41815R1Mechanisms of Adipocyte Regulation: Insights from HADHB Gene ModulationPLOS ONE

Dear Dr. Huang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 16 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Sayed Haidar Abbas Raza

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors should be commended for presenting a much improved article. I am happy for the article to be published

Reviewer #2: This manuscript by Yang et al focuses on the role of the HADHB gene, which encodes the beta-subunit of 3-hydroxy acyl-CoA dehydrogenase, in regulating bovine preadipocyte proliferation and differentiation. The authors have shown that HADHB plays an important role in the regulation of adipocyte proliferation and differentiation, which is a critical process in energy metabolism and lipid synthesis. By applying techniques like siRNA-mediated knockdown, adenoviral overexpression, transcriptomic sequencing, and functional enrichment analysis, the authors identified PPARγ and CEBPα in regulating differentiation. CDK2 and PCNA, were identified as critical for proliferation. Knockdown of HADHB inhibited differentiation, reduced lipid droplet formation, while its overexpression enhanced the processes. Furthermore, knockdown of HADHB promoted early-stage apoptosis in preadipocytes, suggesting its critical role in cell survival. By transcriptomic analysis, the authors pointed out that overexpression of HADHB induced changes in the expression of 283 genes and enrichment of pathways related to lipid metabolism, cell cycle regulation, inflammation, and apoptosis. Protein-protein interaction analysis revealed key genes such as KIF23, SERPING1, and others that are crucial in the regulation of these processes. The manuscript identifies that HADHB modulates the preadipocyte biology by influencing transcriptional profiles and functional pathways, thus its putative role in energy homeostasis and metabolic regulation. The authors propose that further studies should be conducted to determine the signaling pathways and mechanisms through which HADHB mediates such effects, laying a foundation for exploring its role in the regulation of fat traits in livestock and human metabolic disorders. The manuscript is well written. I have a few suggestions to improve the manuscript

1. The authors need to address the limitations of the study.

2. The manuscript does not adequately emphasize the broader implications of the findings. The authors can discuss how the findings could inform strategies for improving livestock productivity.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Mechanisms of Adipocyte Regulation: Insights from HADHB Gene Modulation

PONE-D-24-41815R2

Dear Dr. Huang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager®  and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Sayed Haidar Abbas Raza

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The revised manuscript now looks much improved and the authors have tried to respond to most of my queries raised during the initial review. I have no other queries.

Reviewer #2: The authors have addressed all the concerns. The revised version of the manuscript has taken into account my suggestions.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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