PONE-D-24-15054Unique features of KGN granulosa cells in the regulation of steroidogenic and antioxidant genesPLOS ONE

Dear Dr. Rodgers,

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Birendra Mishra, DVM, 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: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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

Reviewer #3: Yes

Reviewer #4: 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: KGN is a widely used cell line, and my team has also used it multiple times. Thank you for the author's research, which has given me a deeper understanding of the application areas and generalizability of KGN results.

Reviewer #2: The article is sound, generally well written and addresses a relevant subject. There are some minor points deserving attention but, in my opinion, the manuscript can be accepted for publication.

In the introduction, within the lines 85-91, after stating the objectives of the study, there are some comments about methodology, some results and even some conclusions which would be more appropriate elsewhere.

The paragraph within the lines 200-204, describes the statistical model, but it does not specify which responses are analyzed.

In the Results session, for the sake of objectivity, I suggest that some methodological comments made at the beginning of each session should be avoided, since the contents were already explained before.

In the Discussion, it would be expected to start the discussion session emphasizing the most relevant implications to the present study. However, the findings of this study are addressed for the first time in the discussion only in line 321, in the middle of the second paragraph.

In the Conclusion, the authors state that “Depending on which aspect a study focuses on, KGN cells might or might not be a good model for primary granulosa cells…”. The comment about KGN cells possibly being a suitable model for primary granulosa cells may somewhat contradict the conclusion mentioned in the abstract, on which is only emphasized that those may not be a good model.

Reviewer #3: Comments to the author

The study by Tang et al. investigates the unique features of KGN granulosa cells in the regulation of steroidogenic and antioxidant genes. More specifically, the authors using public RNA sequencing data compared KGN cells to human granulosa cells and confirmed that KGN cells had extremely higher expression levels of TXNRD1 compared to human granulosa cells, suggesting that overexpression of TXNRD1 is a unique feature of KGN cells. Next, authors studied expression of steroidogenic genes, antioxidant genes and the response to treatments with cAMP in both KGN cells and KGN cells in which authors knocked down the expression of TXNRD1. Overall, this is an interesting work. However, I have nonetheless identified some points to be clarified or resolved. I believe they should revise the manuscript very carefully.

There are some issues that need to be addressed:

1. Line 98, The dataset GSE193123 is the only human granulosa cell dataset used in the study. The authors should provide a detailed description of this dataset, including the physiological status and other relevant information, in an appropriate section.

2. Line 211, There are only three GEO datasets [GSE161341 (KGN cells), GSE130664 (KGN cells) and GSE193123 (primary human granulosa cells from secondary follicle)] in the Material and Method. Please describe the sources and details of the other three datasets (3 KGN datasets with 3 human granulosa cell datasets).

3. Line 236, The study examines the effects of dbcAMP and forskolin on steroid synthase in KGN cells by assessing gene expression levels, not protein levels. The authors should explain why only gene expression was measured and whether it is a more suitable indicator of steroid synthase activity. Consider including additional experiments to support your conclusions.

4. Line 263, The authors found that the expression of some antioxidant genes changed significantly after steroid production induction, such as the decrease of SOD2. Please discuss whether it is necessary to measure changes in SOD activity.

5. Line 109/113/203/204, The used software version should be specified in detail.

6. Line 425, Gene symbol needs italics.

7. Line 590/597, FDR suggests writing it as a full spelling name.

8. The format and font of the Reference list need to be re-checked and modified by the author.

Reviewer #4: 1、in this study, ACTB was used as housekeeping gene. did you confirm the stability in KGN？

2、in Fig.1, the expression levels of all these genes were significant lower than ACTB, is that suitable?

3、How did you determine the concentration of additives （dbcAMP\\Forskolin）?

4、mRNA expression cannot fully reflect the true changes within cells, it is recommended to supplement protein level testing

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Unique features of KGN granulosa-like tumour cells in the regulation of steroidogenic and antioxidant genes

PONE-D-24-15054R1

Dear Dr. Rodgers,

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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Kind regards,

Birendra Mishra, DVM, PhD

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 #3: All comments have been addressed

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

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

Reviewer #3: Yes

Reviewer #4: 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 #3: Yes

Reviewer #4: Yes

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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 #3: Yes

Reviewer #4: Yes

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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 #3: (No Response)

Reviewer #4: The author made careful revisions based on the suggested changes, so I suggest accept this manuscript and publish online.

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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 #3: No

Reviewer #4: No

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