PONE-D-24-22176Novel and Efficient Plasmid Transfection Protocols for Functional Analysis of Genetic Elements in Human Cardiac FibroblastsPLOS 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 is nice, I have some points that could help improve it:

- The Objective is not very clear. Actually, is clearer in the abstract then in the text. Try to make it more clear in the last few paragraphs of introduction.

- Instead of in the text, make a table with the primers listed in the “methods - RT-qPCR analysis”, and indicate the ones used as housekeeping.

- Its strange the Figure legends are embedded in the Results text.

- If I understood correctly, even after all improvements that actually considerably reduced toxicity and cell death, the actual transfection efficiency was less than 5% for R7 and R12 (indicated by the expression of fluorescent proteins). Please try to make this clearer in the conclusion and in the abstract. The reader should know what to expect in terms of efficiency when applying the improved fine-tailored protocol developed in this study.

- In addition, if the point above is correct (about <5% efficiency), the tittle and abstract actually does not really reflects the results, the tittle says “Novel and Efficient Plasmid Transfection….” I would say the word “Efficient” seems a bit overestimated.

Reviewer #2: The manuscript “Novel and Efficient Plasmid Transfection Protocols for Functional Analysis of Genetic Elements in Human Cardiac Fibroblasts“ provides a valuable contribution to the field of cardiovascular research by presenting a novel and efficient protocol for plasmid transfection in human cardiac fibroblasts. The authors made comprehensive evaluation of transfection conditions and the focus on reducing cell toxicity are notable strengths. However, the manuscript would benefit from addressing the concerns below, enhancing the findings' overall impact.

-For Fig 1B-D, the presentation of data on the percentage of propidium iodide (PI) positive cells is confusing. Unlike Fig1C, the % in Fig1D seems normalized to one of the controls, which demonstrated barely detectable PI+ signals and then made other groups with extremely high (>50~400 fold) changes. Using absolute percentages or normalized data based on the highest group for Fig1C would be reasonable and sufficient to solve this issue.

-‘This suggests that…’, is this a missing statement in the results section of ‘Effect of Plasmid Purification Methods on Fibroblast Viability’.

-Statistical comparisons between groups are missing in Fig 3.

-While the methods are described in detail, including specific volumes, incubation times etc., would be helpful to provide a supplementary table summarizing the optimal conditions for each reagent tested for quick reference.

-As one of the key factors/parameters that affect the efficiency of primary cell transfection, cell confluency is not explored or discussed in the study at all. Different commercial transfection reagents may have varied requirements for optimal gene expression. Meanwhile, the doubling time of HCF cells may also be considered when the actual percentage of transfected cells and cell death are calculated at the examination time point. Did the authors use the same setting for HCF confluency for all transfections and LV transduction? What’s the optimal cell confluency that can further improve the ‘best’ transfection condition identified in the study?

- The manuscript focuses on HCFs derived from a specific source/donor. It would be valuable to test the optimized protocol on HCFs from different donors or sources to ensure broad applicability. Additionally, detailed information, such as catalog number of the human cardiac fibroblast is also missing.

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Reviewer #1: No

Reviewer #2: Yes: Yi Lin

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Novel and Effiective Plasmid Transfection Protocols for Functional Analysis of Genetic Elements in Human Cardiac Fibroblasts

PONE-D-24-22176R1

Dear Dr. Matsuyama,

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.

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Kind regards,

Keisuke Hitachi

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: All comments have been addressed

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

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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 #1: Yes

Reviewer #2: 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 #1: Now the title and abstract seems to better reflect the findings. I can now support publication of the manuscript. Also, all my points were adequately addressed in this revised version of the manuscript.

Reviewer #2: The authors have addressed my concerns raised in the initial review with substantial revisions that significantly improve the manuscript's clarity, rigor, and utility. The enhancements made, such as in data presentation, statistical analysis, and discussion of key experimental parameters, make this work a valuable contribution to the field. I belief that it will serve as an important resource for researchers working with human cardiac fibroblasts.

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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: Gerson D. Keppeke

Reviewer #2: No

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