Fig. 2

PAK2 is a CMS4-specific vulnerability. A. Bubble plot representing outcome of MAGeCK analysis comparing PAK family members in CMS4 to CMS1–3 CRC cell lines. Each bubble represents one kinase, listed randomly. Negative false discovery rate for MAGeCK output was set to 0.01. Bubble size represents calculated median fold change for all sgRNAs targeting the same gene. FC = Fold change. B. 2Log mRNA expression levels for PAK family I members in CRC (primary) cell lines (upper panels) and tumors (lower panels) as determined by microarray or RNA sequencing. Bars indicate mean, error bars represent standard deviation (S.D). C. Western blot for PAK2 protein expression in HT55 & SW948 (CMS2) and HuTu-80 & MDST8 (CMS4). 2,2,2-Trichloroethanol (2,2,2TCE) signal (excerpt taken around 60 kDa region) indicates amount of protein loaded per cell line. Numbers on the left represent molecular weight in kDa according to a stained protein ladder loaded in the gel. Representative blot of N = 3 is displayed. D. Schematic overview of FACS-based co-culture/competition assay in which WT cells were mixed with either doxycycline-induced non-silencing control shRNA or PAK2-targeting shRNA cells at a 30%:70% ratio, respectively. E. Relative contribution of PAK2-KD cells to co-culture in competition assay set-up, over time. Contribution of shRNA-turboRFP expressing cells to population was first normalized to contribution on day 0 post sort, and subsequently calculated relative to contribution of NS shRNA-turboRFP expressing cells at each individual time-point. CMS2 cell lines indicated in blue, CMS4 in green. N = 3, mean and standard error of means (s.e.m., error bars) are plotted. F. Cell confluence measured over time with IncuCyte live-cell imaging, up to 3 days after start of treatment. Values were normalized to the DMSO treated control. CMS2 indicated in blue, CMS4 in green. Representative experiment of N = 3 is displayed, mean and standard error of means (s.e.m., error bars) are plotted