Dear Dr. zhang,

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.

==============================

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Gianpaolo Papaccio, M.D., Ph.D.

Academic Editor

PLOS ONE

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

10.1016/j.apsb.2023.10.014

https://doi.org/10.1002/jcp.30063

https://doi.org/10.1016/j.apsb.2023.10.014

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

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“This study was supported by the Chongqing Natural Science Foundation General Project[CSTB2022NSCQ-MSX0454]”

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

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Additional Editor Comments:

This manuscript investigates the impact of N-glycosylation on NANOG protein and its role in regulating colon cancer stem cell characteristics, proliferation, migration, and apoptosis. The study addresses a relevant topic and the experimental design seems sound in principle. The findings suggest a novel regulatory mechanism for NANOG, a key pluripotency factor in cancer. While the study presents interesting data, the manuscript requires significant revisions to improve clarity, methodological detail, and discussion of limitations. Crucially, the absence of figures makes a comprehensive assessment of the results impossible.

Major Comments:

Title: The current title, "N-glycosylation regulates NANOG affects stem cell characteristics and apoptosis of colon cancer cells," is grammatically awkward, lengthy, and unclear. It should be revised for precision and conciseness.

Missing Figures (Very Critical): All figures (Figure 1 to Figure 8) mentioned throughout the manuscript are absent. Without the visual representation of the data, it is impossible to properly evaluate the results, the statistical significance claims, and the interpretations drawn by the authors. This is a very critical omission. For instance, the verification of molecular weight shifts in NANOG-MUTs (Figure 2B), the quantification of cell activity, proliferation, migration, sphere formation, and apoptosis (Figures 3-8) cannot be assessed.

There is an inconsistency in the manuscript regarding the HCT166/HCT116 cell line. The abstract refers to HCT166, while the Materials and Methods section initially lists HCT116 (CL-0096) and LoVo (CL-0144) but later refers to HCT166 cells in the same section. This must be clarified and consistent throughout the manuscript.

There is a lack of detailed Western Blot Protocol. While Western Blot experiments are mentioned as a method to detect apoptosis-related proteins and verify protein shifts, a dedicated "Western Blot" subsection outlining the detailed protocol (e.g., antibodies used, dilutions, blocking conditions, detection method) is missing from the Materials and Methods. This is essential for reproducibility.

Apoptosis Results Description is ambiguous: In Section 3.6, the statement "the apoptosis levels of HCT116 colon cancer stem cells and LoVo colon cancer stem cells after transfection of 7 MUT plasmids of NANOG protein decreased (Figure 8)" needs careful rephrasing for clarity. If N-glycosylation is stated to "promote apoptosis" (as in the abstract and section title), then the mutation/removal of glycosylation sites (via MUT plasmids) should lead to decreased apoptosis, which aligns with the finding. However, the phrasing "N-glycosylation participates in regulating NANOG protein to promote apoptosis" and then showing decreased apoptosis in mutants can be confusing. Consider phrasing like "N-glycosylation is required for NANOG protein to promote apoptosis" or "N-glycosylation enhances NANOG-mediated apoptosis."

Minor points:

The introduction mentions screening for "N-glycosylase" expression but could benefit from explicitly naming OST-A and OST-B earlier, as these are the specific enzymes identified and discussed in the results.

While the general mutation strategy is stated (N-X-T(S) to A-X-T(S)), more specific information about the exact mutation sites for each of the seven mutants (MUT-1 to MUT-7) would significantly enhance the reproducibility of the study.

Lack of Rationale for CD133+ Cell Selection: why CD133+ cells were selected as the primary stem cell population for colon cancer or provide a relevant citation to support this choice.

Provide additional details or a reference for the "WZ_Camera" software used for measurements, if it is not a widely recognized commercial software.

To address the novelty, the authors should explicitly articulate the unique knowledge gap addressed by their study in the introduction and ensure the discussion clearly differentiates their findings from existing literature on NANOG or N-glycosylation in cancer.

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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.

Dear Dr. zhang,

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 Oct 11 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.

• 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 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,

Gianpaolo Papaccio, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

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 Editor Comments:

The Authors have answered to some critical points but not to a specific one, namely:

3.Lack of Rationale for CD133+ Cell Selection: why CD133+ cells were selected as the

primary stem cell population for colon cancer or provide a relevant citation to support

this choice. Response: Thanks for the suggestions. CD133 (prominin-1) was selected

as a marker for isolating colon cancer stem cells (CSCs) based on extensive prior

evidence demonstrating its association with stemness properties in colorectal cancer.

Multiple studies have shown that CD133+ subpopulations in colon cancer exhibit

enhanced self-renewal capacity, tumorigenicity in xenograft models, and resistance to

chemotherapy—hallmarks of CSCs.( doi:10.3390/ijms24032400; DOI: 10.1186/s13046-025-03308-8 and DOI: 10.1096/fj.12-218222).

This point needs a specific answer as well as that the Authors add citations.

Other point:

The Authors when answering to the critical points write "suggestion" often it is NOT a suggestion BUT a Major point that is critical

[Note: HTML markup is below. Please do not edit.]

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.

N-glycosylation of NANOG regulates stemness and apoptosis in colon cancer cells

PONE-D-25-28463R2

Dear Dr. zhang,

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,

Gianpaolo Papaccio, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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??>

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

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors have convincingly demonstrated that seven N-glycosidic sugar chains (N-glycans) located at the carboxyl terminus of human NANOG play a crucial role in the molecular quality control of the NANOG protein and in maintaining the stem cell properties of colon cancer stem cells. These post-translational modifications further influence the proliferation and migration capacities of colon cancer stem cells.

Overall, this is an interesting and well-executed study that advances our understanding of how N-glycosylation regulates NANOG function, cellular stemness, and potentially tumorigenicity.

The manuscript has been significantly improved following the revisions. The authors have addressed all previous comments in a clear and comprehensive manner, which has considerably strengthened the quality and clarity of the work. The study is well designed, and the experiments have been conducted in an exemplary and rigorous way. The methodology is appropriate, and the results are clearly presented and convincingly discussed. The figures and data are well organized and effectively support the authors’ conclusions.

Overall, this is a well-executed and scientifically sound study that makes a valuable contribution to the field and the revisions have enhanced the manuscript substantially.

Reviewer #2: The aim of the study “N-glycosylation of NANOG regulates stemness and apoptosis in colon cancer cells” is to investigate the functional role of N-glycosylation in the regulation of NANOG protein activity, particularly in maintaining the stemness characteristics and apoptosis of colon cancer stem cells. By identifying and mutating the seven potential N-glycosylation sites at the carboxyl terminus of human NANOG, the study sought to determine how these modifications influence NANOG’s molecular stability, and consequently, the proliferation, migration, and survival of colon cancer stem cells.

The experimental approaches, including molecular cloning, CD133+ stem cell sorting, and functional assays (proliferation, migration, apoptosis), are appropriate and thoroughly performed. The data are coherently presented and adequately support the authors’ conclusions and all the required experiments have been correctly conducted and the revisions effectively addressed the previous reviewers’ concerns.

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Reviewer #1: No

Reviewer #2: No

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