PONE-D-24-28155The CorrelationBetween Cellular O-GlcNAcylation and Sensitivity to O-GlcNAc Inhibitor in Colorectal Cancer CellsPLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: Charoensuksai et al, studied the relation O-GlcNAcylation with colorectal cancer. They measured O-GlcNAcylation, OGA and OGT levels levels in colorectal cancer cell lines and their sensitivity to OGT inhibitor OSML1. They found a dose dependent decline in cell viability in cancer cell lines and Os exhibited a synergistic relationship with regorafenib (Re). They concluded that OSML1 can be a potent anticancer agent when used along with other treatments. It is a well thought study, but the data supporting the claims especially the western blots are low quality. The authors should use proper controls to check the specificity of antibodies especially when measuring O-GlcNAc. Stressing Cells to enhance O-GlcNAcylation can be a good control. I would suggest using Lectins as well for secondary confirmation. Inclusion of one more control line is highly recommended.

Reviewer #2: The manuscript suggests a novel predictive marker, O-GlcNAc transferase inhibitor (OGTi), for O-GlcNAcylation in various cancer cell lines, particularly colorectal cell lines. The manuscript is acceptable after addressing minor grammatical errors and the following comments.

Comments and Suggestions

Background on Regorafenib (Re): The authors should provide a brief background on regorafenib, explaining why it was chosen for the study. Additionally, they should clarify its FDA approval status.

Error Bars and Statistical Analysis: In Figure 2 C, D, and E, the western blot quantification lacks error bars for the intensity of O-GlcNAc, OGA, and OGT in all cell lines. Given that the experiments were performed in triplicate, the authors should calculate the data from all three results, include error bars, and conduct statistical analysis.

Grammatical Error: In paragraph 3.2, "our results evealed..." should be corrected to "our results revealed...".

Flow Cytometry Data: While Figure 6 demonstrates the cytotoxic activity of the combination of Os:Re in NCI-H508 cells, suggesting the efficacy of dual therapy, flow cytometry data is missing for FHC cells (control cells). Including similar flow cytometry experiments for FHC cells would provide valuable insights into the specificity of the treatment.

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Reviewer #1: Yes: Willayat Yousuf Wani

Reviewer #2: Yes: Mohd Tayyab

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The Correlation Between Cellular O-GlcNAcylation and Sensitivity to O-GlcNAc Inhibitor in Colorectal Cancer Cells

PONE-D-24-28155R1

Dear Dr. Charoensukai,

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,

Sadiq Umar

Academic Editor

PLOS ONE