PONE-D-21-35752Differentially expressed microRNAs during the differentiation of muscle-derived stem cells into insulin-producing cells, a promoting role of microRNA-708-5p/STK4 axisPLOS ONE

Dear Dr. Ma,

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.

As you will see, both referees highlight the potential interest of the findings. However, they have raised a number of concerns and suggestions to improve the manuscript, or to strengthen the data and the conclusions drawn. As the reports are below, I will not detail them here, as we think all points need to be addressed. Given the constructive referee comments, we would like to invite you to revise your manuscript with the understanding that both referee concerns must be addressed in the revised manuscript and in a point-by-point response. Acceptance of your manuscript will depend on a positive outcome of a second round of review.

Please submit your revised manuscript by Feb 21 2022 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Abdul Qadir Syed, PhD

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: No

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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: No

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: No

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 objective of the study is well thought and executed.

But there are so many places in the manuscript which need to be improved.

There were no mention of which diabetes was studied here Type I or Type II?

Scientific English need to be used (centrifuging=centrifugation; 150 g=150xg) and reviewed carefully before submission. As line 224 mention: The separate and overlapping DEmiRNAs...... It may be refrain as "Unique and common" DEmiRNAs.... Last line mentioned "Left 11", it can be refrain as "remaining 11".

There are many places where sentences need to be reframed. Few sentences are repeated in Introduction and discussion. Every time Scale bar has been mentioned as separate sentence without making a sentence, which need to be corrected. p-values either be given in sentence or in parentheses.

What is RPMI? Is this a growth medium or Institute?

nictinamide=nicotinamide.

Version and references need to be provided for used Softwares and databases.

Methods: What were the analysis parameters used for processing of Raw sequence data and quality passed data?

Wild-type STK4 (STK4-WT) and the mutant 141 STK4 (STK4-MT) were cloned into pGL3 alkaline luciferase vector. Explain Mutation experiment.

Line 171: performed at least three independent repetitions. Is this assumption or confirmation regarding replicates?

Results:

Sequencing: No result has been mentioned about sequencing (Data obtained, processed in terms of reads and quality). pls provide this detail. What

Line 193: These two markers; Which are these two markers? Explain.

Line 208: Pls provide Figure number for heatmap.

There were 7145 target genes identified but only mention of 1655 for GO term analysis, what about other genes? Pls explain how these GO terms (12 biological process, 6 cellular components and 2 molecular functions) are related to IPCs differentiation.

Venn diagram and heatmap and bar charts are not visible, high quality figures may be submitted.

Line 311: Emerging data suggest that miRNAs act as a pivotal role in IPCs differentiation; "miRNAs play a pivotal role". Refrain the sentence.

Line 315: These researchers; Which researchers referred here?

Line 322: The results showed that these target genes were mainly related to cellular process and signal transduction. "No mention of Which cellular process and signal transduction." Explain and give details.

Line 334: STK4 exerts a regulatory effect on IPCs differentiation via phosphorylating YAP1....... Which kind of regulatory effect "Positive or negative"? Conclusion was based on this comparison and effect was not explained in the manuscript.

Reviewer #2: In the current study, the authors have tried to show the importance and involvement of miR-708-5p during differentiation of MDSCs into insulin producing cells. Although the study is important, experiments needs to be revised properly with proper controls.

1. Most of the experiments involve the expression of proteins by immunofluorescence studies. However, they lack proper control and comparisons. In Fig.1 the MDSCs marker expressions needs to be compared with the 0h cells. Likewise, in Fig.2, the insulin / c-peptide expression can be compared at each stage.

2. Similarly, the experiments involving addition of miR-708-5p mimic needs untreated cells as wells as NC-miRNA as controls.

3. The image quality needs to be improved throughout. The flow plots are not possible to read.

4. Authors need to explain about the STK4-Mut in detail. Is it having a mutated miR-708 binding site? If yes the details needs to be provided.

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6. 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: Yes: Ram Nageena Singh

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.

PONE-D-21-35752R1Differentially expressed microRNAs during the differentiation of muscle-derived stem cells into insulin-producing cells, a promoting role of microRNA-708-5p/STK4 axisPLOS ONE

Dear Dr. Ma,

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.

Specifically, there is still one comment which was not addressed in the revised manuscript. I highly suggest that author should address this comment in the final version of manuscript. 

Please submit your revised manuscript by May 02 2022 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,

Abdul Qadir Syed, PhD

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.

Additional Staff Editor Comments (if provided): PLOS ONE does not provide copyediting or proofs of accepted manuscripts. We therefore recommend that you carefully review your manuscript and correct any language errors at this time.

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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: (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: There is still one comment which need to be addressed.

Statistical Analysis:

Line 182: Each experiment was performed at least three independent repetitions.

This sentence reflects that Authors were confirmed about number of replicates. Why "at least"? Why not that "All the experiments were performed with 3 replicates"?

Pls revise the sentence.

Reviewer #2: The authors have answered all the comments satisfactorily. Now, it can be considered for publication.

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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: Yes: Ram Nageena Singh

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.

Differentially expressed microRNAs during the differentiation of muscle-derived stem cells into insulin-producing cells, a promoting role of microRNA-708-5p/STK4 axis

PONE-D-21-35752R2

Dear Dr. Ma, 

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.

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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,

Abdul Qadir Syed, PhD

Academic Editor

PLOS ONE