Induction of Endoplasmic Reticulum Stress by Prodigiosin in Yeast Saccharomyces cerevisiae
, Thi Hien Trang Nguyen 1, Thi Tuyen Do 1, Thi Thao Nguyen 1, Thanh Hoang Le 1, Thi Anh Tuyet Nguyen 1 and Yukio Kimata 2,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsProdigiosin is a red tripyrrole pigment produced by various bacteria and it has been investigated in current report. I like to give the following comments.
1. In the introduction, line 63, prodigiosin inhibits protein folding in the ER, leading to UPR evocation. What is the merit of this result?
2. In line 57, the purity of Prodigiosin was unknown.
3. In line 100, two different HAC1 primer sets used in RT-PCR must show in clear.
4. In Figure 1, legends need to explain in detail. It is hard to follow the main meaning of Prodigiosin activates the Ire1/HAC1-dependent UPR signaling pathway. Sample size in each group shown in column was unknown. Dose-dependent effect of Prodigiosin was not observed in (A). Percentage of what shown in (B) remained obscure. Please check the data carefully.
5. In Figure 2, what is the stress used in this screening? Quantification of the experimental results is extremely required. Please explain the details in legends.
6. Table 1 needs the legends to explain in detail. What is the sample size in each group? Statistical difference shall be compared in clear.
7. Please rephrase the discussion section. It failed to reach the standards.
8. Conclusion is required. Novelty and limitation of current report will be helpful.
Comments on the Quality of English LanguageIt seems better to check through a professional editing.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
thank you for providing this manuscript. It is written in good Englisch, easy to follow and good to read.
I have only a few suggestions:
Line 16: please put Saccharomyces (S.) cerevisiae to italic letters
Please make sure you introduced all abbreviations (Ire, ATF, BiP ...).
Yeast culturing: Did you do any tests to confirm identity of cells before your assays?
Line 109: Please explain which software you used for measuring the inhibition zone.
Figure 2: Could you please provide a higher resolution of this pictures? Please add A, B, C.
Thank you!
kind regards
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript describes experiments in budding yeast testing whether the molecule Prodigiosin induces the unfolded protein response (UPR). Evidence from the literature pointed to induction of the ER stress response in mammalian cancer cells, providing a sound rationale for the experiments here. Testing UPR induction in yeast is straightforward, and the experiments carried out by the authors are appropriate to test the hypothesis. The data are clear and appropriately interpreted. I have two suggestions to improve the manuscript:
(1) I think it would be important to show the gels with the Hac1 splicing results, not just the quantification. I also think it would be helpful to show representative images of the inhibition circle assays, not just the quantification.
(2) A major question that arises from these new data and the existing data is whether the ER stress/UPR is a symptom of the major inhibitory/antibiotic effect of Prodigiosin, or whether some other cellular effect is responsible for inhibiting growth. One straightforward way to address this question would be to attempt to isolate yeast mutants that are resistant to concentrations of Prodigiosin that block colony formation. (Perhaps this has already been done.) Then, simply ask whether these resistant mutants still show evidence of UPR induction. If so, then UPR induction/ER stress is not the major cause of growth inhibition. If not, then it is possible that UPR induction/ER stress is the major cause of growth inhibition; it is also possible that the resistant mutant simply doesn't uptake as much of the Prodigiosin, for example. It would not be necessary to perform further analysis, in my opinion. If the authors are unable to isolate a Prodigiosin-resistant mutant, then obviously this experiment cannot be performed, but I think it would be worth trying.
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsIt has been revised in a good way.
Comments on the Quality of English LanguageIt seems better to check through a professional editing service.
Reviewer 3 Report
Comments and Suggestions for AuthorsI am satisfied by the changes made in the revised manuscript to address the concerns with the original manuscript.
