Figure 4: ROCK–LIMK–cofilin axis is required for WSS-induced YAP1 activity.

(a) Cofilin, gelsolin and YAP1 protein levels were measured in stable PC3 lines generated by lentiviral shRNA. (b) Gelsolin knockdown did not significantly change movement speed, but silencing of cofilin increased velocities of cells cultured under WSS as well as static conditions (n=3 independent experiments, Kruskal–Wallis One-way ANOVA, **P<0.001; multiple comparison of individual group was tested by Dunn’s method, *P<0.05). (c) Enhanced motility in cofilin knockdown cells in static and WSS conditions was significantly reduced by YAP ablation (n=3 independent experiments, Kruskal–Wallis One-Way ANOVA, **P<0.001). (d,e) LIMK inhibitor (LIMKi3) elevates levels of phosphorylated YAP1 and blocks motility (n=3, Kruskal–Wallis One-way ANOVA, **P<0.001). (f) Treatment with the ROCK inhibitor Y27632 elevated levels of phosphorylated S127 YAP1 present in WSS-treated PC3 cells, suggesting that ROCK normally plays an important role in promoting dephosphorylation of YAP1 to interrupt its sequestration in the cytoplasm. (g) WSS-induced motility is blocked by inhibition of ROCK (unpaired t-test, **P<0.0001). (h) WSS increases FAK phosphorylation at Y397 in PC3 cells at 30 min. The PF-562271 FAK inhibitor reduces phosphorylation of FAK and increases phosphorylation of YAP1. (i) Representative images of YAP1 subcellular localization at 30 min of static or WSS culture. Cells in suspension express negligible amounts of pFAK. (j) Inhibition of FAK reduces YAP1 nuclear localization in static cultures (unpaired t-test, **P=0.002) and, to a lesser extent, in WSS cultures (unpaired t-test, P=0.07). Decrease in YAP1 nuclear localization differed significantly between static and WSS when normalized to vehicle controls (unpaired t-test, *P=0.02), suggesting that WSS can act via FAK-independent mechanisms to stimulate YAP1. Error bars represent±s.e.m.