The article entitled “Identification and targeting of a HES1-YAP1-CDKN1C axis in fusion-negative rhabdomyosarcoma” by Kovach AR et al. describes the crosstalk between Notch and Hippo pathways in fusion negative rhabdomyosarcoma (FN-RMS). The authors performed both genetic and pharmacologic inhibition of the Notch effector HES1 and analysed the related effects on FN-RMS in vitro and in vivo models.
The authors demonstrated that HES1 inhibition affect cell growth and induce myogenic differentiation by CDKN1C up-regulation. It is not clear, at least at this time, the molecular mechanism of YAP1 and HES1 on CDKN1C regulation.
The research design is good but could be improved. The results are not well described neither in the main text nor in the figure legends. The conclusions are supported by the results. Below are listed some points that need to be addressed:

Major points:
1) Honestly HES1 protein levels showed by WB in SMS-CTR and Rh36 cells in Figure 1B do not reflect the fold changes obtained by real-time PCR reported in figure 1A. Moreover, Actin B levels, especially in SMS-CTR and Rh36 cells, suggest that the gels are not normalized. These blots need to be changed.
2) Since the terminal differentiation was confirmed in RD but not in SMS-CTR cells and considering that HES1 depleted Rh36 cells exhibited more elongated cellular bodies (Figure 1D), I suggest the authors to analyse the expression levels of myogenic markers by real time and western blotting assays in Rh36 cells in order to be able to draw general conclusions.
3) The expression of some apoptotic markers as Bcl-2, cleaved PARP and cleaved caspase 3 should be analysed by Western blotting to better understand the apoptotic process induced by HES1 silencing.
4) It could be interesting to analyse the senescence markers p16 and p21.
5) Figure S3, despite the strong down-regulation of both HES1 and YAP1, does not show CDKN1C increase after treatment with inducible Hes1 shRNA as stated by the authors in line 315. The text should reflect this result. Do you have any explanation?
6) YAP1 overexpression does not reduce HES1 transcript levels (Figure S8), whilst in a previous work the authors described HES1 transcript levels down-regulation after YAP1 silencing (Slemmons et al., 2015). What is the possible reason proposed by the authors? Please, add some explanation in the discussion section.
7) The authors showed CDKN1C up-regulation after both HES1 (Figure 2) and YAP1 (Figure S1C) knockdown. I think it is necessary to analyse the expression levels of CDKN1C in depleted HES1 RMS cells overexpressing JAP1 and in silenced JAP1 RMS cells overexpressing HES1 to better understand the relationship between HES1, YAP1 and CDKN1C in FN-RMS.
8) Please check the English language throughout the paper, some sentences in the main text and in the figure legends are incorrect.

Minor points:
1) How many times the experiments were performed? This information needs to be clearly specified in the “Material and Methods” section.
2) line 148 in “Immunoblotting” section: remove anti-cleaved PARP (Cell signaling #5625,1:1000) since it is not present in the western blotting showed in the manuscript and add Actin B, with the relative abbreviation.
3) I think that the sentence “in 5% w/v sucrose or 5% w/v sucrose (control)” in line 177 is incorrect. Please, check it out.
4) line 186: after 10 days of treatment.
5) line 188: you wrote “pharmacologic” twice.
6) line 193: after 14 days of treatment.
7) line 200: you mean “identified”?
8) line 214: 200μM or μl? Please, check it out.
9) Figure 1A-B and Figure 5A: RD are in the left not top panel and Rh36 in the right not bottom panel.
10) Please specified the magnification and the scale bar of the following figures: Figure 1D, Figure 3C-F, Figure S2C-E, and Figure S4D.
11) The legend of Figure 4A-B is not correct. Please control all the figure captions.
12) line 342: IC50 is reported only in Figure 5A. Please describe Figure 5B correctly.
13) Figure S5A and C are not mentioned/described in the main text.
14): Panel D and E of Figure 5 and Figure S5 are not described in the figure legends.
15): Panel C, D and E of Figure S7 are not properly described in the figure legend.

Kovach AR et al. have submitted a revised version of their manuscript after many months, nevertheless some major points raised by the reviewers have not been addressed.
Specifically, the conclusions on myogenic differentiation and apoptosis are not supported by the results yet, and more importantly the relationship between HES1, YAP1 and CDKN1C, which is the major goal of this manuscript, has not been deeply analysed.
Major revisions need to be performed to support the observations made so far.

1) The authors performed HES1 silencing in 3 models of ERMS (RD, SMS CTR and Rh36). Based on the western blot showed in Figure 1B, the transfection efficiency is confirmed only in RD cells, which indeed showed significant increase of myogenic markers at both mRNA and protein levels. However, the induction of myogenic differentiation was not confirmed in SMS CTR cells and was not analysed in Rh36 as previously requested. With these incomplete results the authors cannot state that HES1 silencing induces myogenic differentiation in RMS cells.

2) As concern the apoptosis, the authors performed new western blot to analyse the expression levels of cleaved PARP and cleaved caspase 3, but they detected only the full length of these proteins. I suggest i) to use a positive control to exclude that the antibodies are not working well and ii) to perform a time course to find the correct timing of the apoptotic response. Finally, since Caspase 3/7 luminescence assay showed strong activity, caspase 7 could be analyse by western blot to demonstrate that the apoptotic process is caspase 3-independent. I suggest to include the results showed in Figure R3 (for review not publication) in the main manuscript (p16 excluded).

3) As stated before, the expression levels of CDKN1C in depleted HES1 RMS cells overexpressing JAP1 and in silenced JAP1 RMS cells overexpressing HES1 have to been shown.