Inhibition of Hedgehog Delays Liver Regeneration through Disrupting the Cell Cycle
Round 1
Reviewer 1 Report
1. In figure 5 A and B, there is no significant statistical difference between 2 groups in mRNA level but does have difference in protein level, authors need to explain the results and why authors decide to proceed Western blots even there is no difference in mRNA expression
Author Response
Q: In figure 5 A and B, there is no significant statistical difference between 2 groups in mRNA level but does have difference in protein level, authors need to explain the results and why authors decide to proceed Western blots even there is no difference in mRNA expression
Answer: Thank you for your questions. The phenotypes of mice experiment showed that the liver regeneration and cell proliferation slowed in the vismodegib-treated group, then our original plan was to fully examine the changes associated with cell proliferation and cell cycle in mRNA and protein levels. Hence, we detected the protein level of Cyclin B via western blots, though there was no significant difference in mRNA level. The changes of Cyclin B in protein levels were unexpected and inconsistent with the change trend of mRNA. We speculated that Cyclin B was not regulated by Hh signaling pathway in transcriptional level, but there was protein level regulation that affected the changes of Cyclin B. Whereafter we detected the related proteins (BUB1B and CDC20) that regulate the degradation of Cyclin B, which showed the trend with expectations. Therefore, we concluded that inhibition of Hh signaling pathway did not affect the changes of Cyclin B mRNA, but inhibited the degradation of Cyclin B at the protein level, resulting in the accumulation of Cyclin B.
Reviewer 2 Report
The manuscript by Tao et al 2021 provides an insight into the role of hedgehog activity and signaling in the process of liver regeneration. Prior to publication the authors will need to address the following:
Manuscript needs a thorough spelling and grammar check from start to finish, including title and abstract. Suggest an appropriate agency as the manuscript is replete with errors that in parts hinders the understanding of the text.
All acronyms should be defined at first usage e.g ICR, RIPA, SDS etc.
Check the writing format such as 50 of IC50 should be subscripted etc.
Authors should provide details of the ethics approval and associated study number.
Was a paired student t-test used as stated for the stats, in which case was the data normally distributed, it doesn’t look to be?!
Section 3.1
Why is the relative protein expression of Gli-3 not included in Figure 1B, presumably there are no statistically significant changes? And the mRNA changes are also minimal. It is useful to include since further analysis of Gli-3 is not undertaken. It might be more informative to the reader if the plots of Gli-1, Gli-2 and Gli-3 are included on the same figure and therefore the same y-axis of fold expression is included. Likewise for the protein, since these are minimal changes at the protein level.
Figure 1C is missing a scale bar on the images and secondly to state that the IHH and SMO expression was significantly increased (2 days post-PH) in the text of the document, this must be supported with appropriate quantitation and provision of histograms or similar that quantify these changes and that this is undertaken with similar regions.
Section 3.2 – provide references that supports the statement that liver to body-weight ratios remain constant (and for which age of mice) and that repair to normal liver mass is within approximately 10 days. The authors mention that this was the case for their studies but do not show any data, this could be included as supplementary data. Similarly, it would be useful to visualize the reduced liver regeneration process that the authors mention after treatment with vismodegib, and this could be included via histological sections of the liver.
Add a line or two (with a reference) to better explain the basis of the Edu proliferation assay (Figure 2C) and list figures in numerical and alphabetical order, such that Figure 2B should be listed before 2C. For Figure 2B, are any of the differences in body weight significant, if so, provide asterisks on the Figure, but if not, then mention this in the text of the document. For Figure 2D, did the authors consider any pre-hepatic or post-hepatic liver damage, and therefore expand the list of liver biomarkers, such as including bilirubin (total and conjugated) (pre-hepatic) and ALP and GGT (post-hepatic) damage?
Section 3.3. Unless I have misunderstood, the control data of Figure 4A and B are the same experiments performed in Figure 1, but perhaps with additional mice? I appreciate that the story of this figure is to consider the effects of vismodegib but Figures should not include data repetition. For Figure 3C, as above, no scale bars included and no quantitation performed.
Figure 3.4 – the analysis is first with cyclin D and then Cdk2 (Figure 4, part A), the blot that follows should be in the same order unless the blot has been loaded to reflect molecular weights, in which case they should be added to the figure. The quantitation of the western blots (Figure 4b) is incomplete, it is ok to provide numbers, but this should be shown as histograms as with the other Figures and therefore provide information as to whether any of the changes are significant. Figure 4C, include scale bars and could the authors not provide an image from flow cytometry of the different fractions of dividing and non-dividing cells? (flow cytometry is used in Figure 5).
Section 3.5, Figure 5B not presented as a histogram with details of significant changes not included. Figure 5C needs to be in higher resolution and ideally larger such that the writing on the figure is legible. Figure 5D include scale bars.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 2 Report
The authors have done a good job in addressing the requested review and I am happy to recommend the publication of the revised manuscript.
