PONE-D-25-27690

Study on the Mechanism by which NFIC Inhibits the Development of Glioma by Regulating the SHP2/PI3K Signaling Pathway through the NF-κB/PTEN Signaling Regulation

PLOS ONE

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Dear Reviewer, Thank you for pointing out that the manuscript, including file naming conventions, should comply with PLOS ONE formatting requirements. We have revised the manuscript according to the PLOS ONE format template and have incorporated these changes into the revised version. We sincerely appreciate your thorough review.

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Dear Reviewer,

Thank you for pointing out the necessity to provide all original images used for blot or gel experiments. We have now annotated the blot/gel image data according to the requirements for blot/gel reporting and figure preparation. The updated images have been incorporated into the revised manuscript. We sincerely appreciate your meticulous review.

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Dear Reviewer,Thank you for noting the absence of an ethics statement in the Methods section of the manuscript. We have supplemented the Methods section with an ethics statement as requested. The updated data are now incorporated into the revised manuscript. We sincerely appreciate your thorough review.

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Dear Reviewer: Thank you for pointing out that all PLOS journals now require that all data underlying the research results described in their papers be freely accessible to other researchers. We have supplemented the open data as requested. The updated data are now incorporated into the revised manuscript. We sincerely appreciate your thorough review.

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Dear Reviewer: Thank you for noting the absence of individual captions for the figures. We have reintroduced separate captions to enhance readability. The updated figures are now incorporated into the revised manuscript. We sincerely appreciate your thorough review.

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Dear Reviewer, Thank you for pointing out the missing citations for Figures 1 and 4. We have reinserted the citations for Figures 1 and 4 to enhance readability. The updated citations have been incorporated into the revised manuscript. We sincerely appreciate your meticulous review.

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Dear Reviewer: Thank you for pointing out the issue with the updated references. We have replaced the original references with appropriate literature supporting this pathway. As previously mentioned, we have also supplemented relevant literature and included corresponding data and results. Once again, we sincerely appreciate your meticulous review.

We sincerely appreciate your meticulous review and professional guidance.

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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

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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

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Reviewer #1: No

Reviewer #2: Yes

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: Study on the Mechanism by which NFIC Inhibits the Development of Glioma by Regulating the SHP2/PI3K Signaling Pathway through the NF-κB/PTEN Signaling Regulation. This article has a clear research framework, but there is still important room for expansion in depth, breadth of verification and mechanism interpretation.

1.There are duplications in logical statements. Paragraphs like "NFIC promotes the expression of OGN and PTEN..." are repeated three times in the text. Redundant content is simplified and terminology is used in a unified way, such as "NFIC high expression group is unified as NFIC-OE."

Dear Reviewer, thank you for pointing out the redundant logical statements in the text. We have standardized terminology throughout the manuscript, replacing phrases like “NFIC promotes the expression of OGN and PTEN...” with “The NFIC high expression group is unified as NFIC-OE.” We have also confirmed that all other formatting requirements comply with journal standards.

2. Whether OGN, PTEN, and SHP2 are the "necessary paths" for the NFIC effect requires additional experimental verification to improve the core mechanism closed loop.

Dear Reviewer, thank you for noting that whether OGN, PTEN, and SHP2 constitute “essential pathways” for NFIC effects requires additional experimental validation. We have supplemented the revised manuscript with Western blot analysis from co-immunoprecipitation (CO-IP) experiments and clinical trial data. All these experiments and their results are now fully incorporated. We appreciate your thorough review once again.

3.The "Bioinformatics Analysis" in the results section does not indicate the content shown in Figure 1; the naming and layout of the figures and tables do not fully conform to the PLOS ONE format, and the legends are missing. Further standardize the layout of the figures and tables and the writing of the legends.

Dear Reviewer: We appreciate your observation that the “Bioinformatics Analysis” section in the Results did not display the content of Figure 1, lacked sufficient figure captions, and omitted legends. We have comprehensively revised all figure captions to clearly indicate sample sizes, number of independent experiments, statistical methods, and added p-values and significance indicators to each figure. We sincerely thank you for your meticulous review and valuable suggestions.

4.Western blot experiments are sufficient, but they rely too much on WB experimental results, resulting in the analysis of the results being mainly described as "up-regulated genes" and "down-regulated genes", which is general and lacks rigor. Quantitative statistical analysis of WB experiments can be further added to make the results more statistically significant.

Dear Reviewer: Thank you for pointing out that the WB results description was overly general and lacked rigor. We have revised the results description in this section, regenerated all figures using correct and independent data, and updated all relevant data in the revised manuscript. We appreciate your thorough review once again.

5.The study has limitations such as a single data source and lack of clinical data verification. How can these problems be solved in subsequent studies to more deeply explore the role and mechanism of the NFIC regulatory axis in human glioma patients?

Dear Reviewer: Thank you for highlighting the study's limitations, including reliance on a single data source and lack of clinical validation. As previously mentioned, we have incorporated CO-IP experiments and clinical trials to further explore the role and mechanisms of the NFIC regulatory axis in human glioma patients. Relevant data and results have been added to the revised manuscript. We appreciate your thorough review.

6. The research methods and background introduction of the article also need more literature support. Please refer to the relevant literature: BIO Integration (2 articles) DOI 10.15212/bioi-2022-0014, 10.15212/bioi-2023-0013; World J Emerg Med (1 article): PMID 37969224; Research (Wash DC) (2 articles) PMID 37040507, 35474903.

Dear Reviewer: Thank you for highlighting the need for additional literature support in the study methods and background section. We have replaced the original references with appropriate literature supporting this pathway. As previously mentioned, we have also supplemented relevant literature and included corresponding data and results. We appreciate your thorough review once again.

Reviewer #2: This article focuses on the tumor suppressor mechanism of the transcription factor NFIC in glioma, attempting to negatively regulate the proliferation and invasion of tumor cells by up-regulating OGN and PTEN and thereby inhibiting the NF-κB/SHP2/PI3K/AKT signaling axis. Overall, the topic selection has certain scientific significance, the experimental methods are relatively complete, and the preliminary results support that NFIC may be a potential therapeutic target. However, the manuscript still has many problems in terms of logical structure, mechanism verification, integrity of experimental design and writing norms. It is suggested that the author make careful revisions based on the following specific opinions to further improve the quality and persuasiveness of the research.

1.The title is too long and the logic is complex. It is suggested to simplify the title into a description of the key mechanisms under the framework of "dual-path regulation"

Dear Reviewer: Thank you for noting that the title was overly long and logically complex. We have simplified the title as suggested to describe the key mechanism within the “dual pathway regulation” framework. Once again, thank you for your meticulous review and guidance.

2.The introduction

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