Fig. 4

PAK2 is essential for attachment signaling and orchestrates actin cytoskeleton remodeling and focal adhesion localization. A, B. Representative images (A) and quantification (B) of crystal violet-stained clonogenic assays with cells expressing non-silencing or ARHGEF7/βPIX targeting shRNAs. N = 3, mean ± standard deviation are plotted, N.S. = not significant, ** = P < 0.01 as calculated in two-tailed, Welch-corrected Student’s T-test. C. Stainings for tubulin, vimentin and actin (phalloidin) 24 hours post-plating. Region of interest (ROI) magnified for actin staining indicated in white dashed box. Scale bar represents 20 μm, ROI images scale bar represents 10 μm. D, E. Stills from time-lapse TIRF microscopy at t = 0 of cell lines transduced with Lifeact-GFP (green) and paxillin-mCherry (magenta). Region of interest (ROI) selected is outlined by white dashed box (D). White dashed line in ROI images indicates region selected for kymographs (E). Scale bar left images represent 20 μm, ROI images 5 μm. See corresponding movies “Supplementary Movies 1, 2, 3, 4, 5 and 6”, available online for the full time-lapse recording. F. Representative western blot images showing phosphorylated cofilin (Ser3) and total cofilin in PAK2 proficient (WT) and deficient (KO) cells. Numbers on the right represent molecular weight in kDa according to a stained protein ladder loaded in the gel. Throughout the figure CMS2 is indicated in blue, CMS4 in green