In the submitted manuscript, Schelch and colleagues investigate MPM and the contribution of YB-1 in its proliferation and migration. MPM, as all mesothelioma types, warrants more research effort in the community, and the authors deserve applause for tackling this challenging tumor in their work.

The manuscript bases on a cell-based overexpression approach to study YB-1's impact on one MPM cell culture model. The authors uncover that while YB-1 overexpression (stated to be at the protein level even below levels achieved in certain MPM tumors) increases proliferation somewhat, the major impact on MPM cells is the increased migratory capacity. The authors document this effect in several assays. A technical feat of the manuscript is the use of CDS vs 3'UTR targeting siRNAs in their culture systems, which enables more fine-grained level control - and that the authors nicely document in their blot.

Mechanistically, the authors link YB-1 activity to the altered gene expression and protein levels of several known downstream target genes, and predominantly to EGFR and SNAI1. This aspect of the manuscript appears suddenly and has little in terms of previous intro or rationale, with especially SNAI1 appearing like a late after-thought in the narrative. This aspect of the paper would benefit from a re-write and revisiting the rationale, as outlined below.

Overall, the authors provide an elegant MPM-focused model to study YB-1 function and contribution to MPM biology. However, the authors should revisit individual statements throughout their manuscript to place their work in better context (see below). Once revisited, the work will certainly be of utility and interest to the mesothelioma community and beyond.

• Major points:
  1) The authors nicely quantify the overexpression of their initial transgene and provide further quantifications of YB-1 levels. Throughout, however, the comparison to the situation in different MPM tumors becomes blurry, i.e., how do the levels of each of the authors' overexpression systems compare to native tumors? Providing comparative data in a sentence or two at relevant passages throughout the text would greatly help with this assessment and would provide context for the authors' work and findings. Similarly, the authors should make a clear statement as to how their initial, constitutive YB-1 transgene compares to their Dox-controlled versions (and their prior work on miR-137).

2) The authors use xenotransplants in zebrafish as in vivo proxy for cell migration and tissue invasion. However, the presented data could also simply be interpreted as Dox-treated tumor cells (or embryos) having facilitated transport of the injected cells in the blood stream. The authors state that their data reveals crossing of vessel walls by the injected cells; yet, the images and data provided do not allow the reader to assess this - the observed accumulation at the tail end could also be simple physical trapping in the narrow vessels of the caudal hematopoietic territory. Can the authors provide higher resolution images of the CHT or tail region to assess tissue invasion or overall localization of the MPM cells?

3) The authors select several genes that have been linked to YB-1 control, and ultimately end up focusing on EGFR and SNAI1. As outlined above, this focus is not well rationalized and comes suddenly, giving the narrative and data a sense of preliminary findings. The authors are encouraged to introduce transcriptional vs translational control of target genes by YB-1 in their intro, including SNAI1.

• Minor points:
  a) The authors establish that EGFR is upregulated by YB-1 overexpression and test pharmacological interference with its activity. Can the authors discuss if the effect of increased EGFR is EGF ligand-dependent (i.e., do MPM cells generate EGF, does the medium contain it, etc.) or if the authors think this could be due to ligand-independent activation of the RTK by increased protein levels? This might be an influential point concerning how EGFR is considered as therapeutic or diagnostic/predictive target.

b) As by far not all MPM tumors feature significant YB-1 upregulation, and with mesothelioma being inherently heterogeneous, the authors should place their work on YB-1 in overall context of mesothelioma biology, i.e., that YB-1 could provide a stratifying marker in a sub-set of tumors.

The authors are to be commended for their thorough revisiting of their initial submission and for addressing the reviewer comments in a thorough manner.

The now revised version of the manuscript seems much more accessible to the reader and in terms of rationale. Besides a few typos here and there (e.g., Figure S5 top-right panel), some of the newly added text at the end of sections (such as at 3.4) would benefit from a concluding summary sentence of the findings.

Otherwise, the work will be of interest to the mesothelioma community and hopefully stimulates future work on YB-1 in the field.