Peer review of: The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells (2020)



Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This is a revised manuscript that examines the relative roles of CDC25, NIP30, and REGγ on 
proteasome-dependent degradation of p21 and consequent cell growth. As with the previous 
version of the manuscript, the results allow the authors to present a model whereby NIP30 
regulates REGγ activated proteasome activity via CDC25 control of NIP30 phosphorylation status. 
This is an interesting and potentially important set of results. 

The authors have responded to my original concerns to improve the manuscript. However, in my 
opinion, their responses are not completely satisfactory and in most cases consist of minor 
additions to a largely unchanged text. For example, I thought that reference to previous work by 
others was not sufficiently acknowledged in the setup to this work and thought that a satisfactory 
response would include a more serious reworking of the introduction and first part of the results. 
Instead the authors have simply added references in several places. 

As noted in my original comments, I continue to think that a major unresolved issue here is the 
molecular mechanism by which REG gamma/NIP30 regulates proteasome action against protein 
substrates such as p21 and suggested an in vitro experiment that could directly examine this 
process and test the authors’ models. The authors have provided such an experiment 
(Supplemental Figure 3F), but it is incompletely described and therefore difficult to evaluate; there 
is no indication of relative concentrations of proteins in the assay, details of the incubation 
conditions, validation that the single time point is an appropriate measure of rate, etc. The use of 
in vitro translated (in what system?) p21 (instead of bacterially expressed and purified p21) 
introduces lysates into the system. Maybe it makes no difference, but who knows? The single p21-
only “control” is not sufficient without a detailed description of the assay conditions and 
components. 

The model figure (6E) is somewhat confusing and perhaps misleading since there is no direct 
experimental evidence to show whether the effect of CDC25 action on NIP30 phosphorylation 
affects proteasome activity by blocking REGγ/NIP30 interaction in isolation or whether this occurs 
within the proteasome complex. This is an issue that could add considerable mechanistic detail to 
the work and could be tested directly, but is not done so here. 

Blanket statements in many figure legends, such as “All experiments were repeated three times.” 
are not entirely clear. Does that mean the complete xenograph experiment shown in Figure 6C and 
6D was done independently three times? If so, why not combine all of the data? 

The presentation is improved but there are still many examples of grammatical errors and odd 
syntax throughout. 

Reviewer #2 (Remarks to the Author): 

My major concerns have been addressed. However, although the grammar and word usage have 
been much improved, the text is still rough. Also, the figures are still very small and very difficult 
to read. 

Reviewer #3 (Remarks to the Author): 

The resubmitted manuscript is much improved and addresses the majority of problems in the first 
submission. The major claims are substantiated and present new evidence regarding tumor 

suppressor and p53-independent functions. 

A few problems with wording remain in the Discussion: lines 495, 498, 527 and 542. 

Overall, a much improved submission.