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This review reflects comments and contributions from Teena Bajaj, Arpita Ghosh, Rachel Lau, Garima Jain, Femi Arogundade, Marta Oliva-Santiago & Isaac Arddey. Review synthesized by Garima Jain & Arpita Ghosh.

Overall, the research article titled "S-Nitrosylation of Transforming Growth Factor β (TGFβ) as a Novel Mechanism of TGFβ Regulation" presents an interesting investigation into the role of S-nitrosylation (SNO) of TGFβ in breast cancer progression. The study attempts to elucidate the impact of NO-mediated modifications on TGFβ activity, dimerization, and protein-protein interactions. The significance statement is well-written and highlights the potential implications of the study's findings.

General Comments

Positive Aspects of the Paper

• The study touches upon the potential implications of reduced S-nitrosylation in the context of health and disease, such as its link to cancer progression and fibrogenic signaling. This makes it relevant to understanding disease mechanisms and potential therapeutic interventions.

• The study employs a variety of techniques, including co-immunoprecipitation, immunofluorescence, ELISA, and molecular biology methods. This multi-pronged approach enhances the robustness of the findings.

• The study includes numerous figures and data, which are helpful for readers to understand the experimental results. This detailed presentation of results aids in transparency and reproducibility.

• The study goes a step further by delving into structural analysis, predicting 3D monomeric and dimeric structures of TGFβ1. This provides insights into the potential structural changes caused by S-nitrosylation.

• The study addresses an important topic in cancer research by investigating the role of S-nitrosylation in TGFβ1 activity, which is known to play a pivotal role in tumorigenesis and fibrogenesis.

• By linking TGFβ1 activity to NO levels, the study contributes to the understanding of NO biology and its implications in cellular signaling.

Major Comments

• The paper is nice in showing the effects of nitric oxide on TGF-beta activity but it requires some checking on consistency. These include (1) the use of CA1d in only assessing TGFBR1 activation but not on p-/T-SMAD3 and TGF-beta levels with modulating NO levels, (2) LTBP in figures 1 and 2 but not addressed in the results, and (3) the use of 293FT cells in the methods but not mentioned anywhere in the results. 

• It would be helpful to briefly mention the broader context of S-nitrosylation and its relevance in cellular signaling and TGFb biogenesis to provide more context for readers who may not be familiar with this topic.

•  It would be beneficial to explicitly state the hypothesis being tested in the present research.

• Discuss the limitations of the study, such as the limited use of malignant cell line (Some of the the data is not available for malignant cell line, hence not reflecting the effect of NO on TGFb in malignant state) and the need for further validation in animal models or clinical samples.

Minor Comments:

• The paper can be improved with some clarity on some figures/analysis. These include (1) scale bar on the immunofluorescence images, (2) addressing whether galunisertib treated and non-treated samples were run on the same gel or on separate gels and (3) the parameters on predicting the potential S-nitrosylation sites and the 3D monomeric and dimeric structures of TGFB1.

• The study primarily focuses on short-term effects of altered S-nitrosylation on TGFβ1. Investigating the long-term consequences and stability of these changes is important, particularly in the context of chronic conditions.

• The study primarily relies on in vitro experiments using cell lines. To validate the relevance of these findings in the context of actual disease progression and breast tissue, in vivo studies using animal models or clinical data are needed.

• The manuscript is generally well-written, but there are some instances where the wording could be clarified for better understanding.

• Care should be taken to avoid repetitive phrases or sentences. For example, there is a repeated use of "SNO-defective" and "SNO-mimetic" without clear definitions or context.

Comments on Statistical Analysis

• The statistical analysis of the data is robust, and the presentation of results with standard error of the mean (SEM) adds transparency to the reported findings.

Suggestions for Future Studies

• X-ray crystallography of S- nitrosylation-mimetic/defective TGF-beta

• Conduct a comprehensive proteomic analysis to identify other proteins that are S-nitrosylated in response to NO levels. This could reveal additional pathways and mechanisms by which NO regulates cellular processes.

• Explore the role of S-nitrosylation of TGFβ1 in other disease contexts beyond breast cancer, such as lung fibrosis, liver fibrosis, or cardiovascular diseases.

• Examine how the S-nitrosylation of TGFβ1 interacts with other signaling pathways, such as the TGFβ/Smad pathway, Wnt signaling, or PI3K/Akt signaling, to understand the broader network of signaling involved in these processes.

• Investigate the clinical relevance of the findings by conducting in vivo studies, such as animal models or clinical trials, to validate the therapeutic potential of modulating S-nitrosylation of TGFβ1 in cancer or fibrotic diseases

Limitations:

In scientific research, it is recognized that all studies possess inherent limitations. Therefore, it would be beneficial if the study authors allocate a dedicated section to systematically discuss these limitations. This section should encompass potential sources of bias, constraints encountered in the experimental setup, and any other factors that could impact the study's outcomes and interpretations. Such transparency enhances the overall quality and credibility of the research.

Competing interests

The author declares that they have no competing interests.