Fig. 2

SEMA6A depletion reduces RhoA activity and induces YAP phosphorylation and cytoplasmic retention in BRAF-mut melanoma cells. A: western blot (WB) analysis of SEMA6A, phosphorylated and total YAP was performed on total cell extracts from inducible shCtrl and shSEMA6A A3 and H2 2/59 cells upon silencing induction. The anti-tubulin antibody was used to validate equivalent amount of loaded proteins in each lane. B: Fold change number of viable A3 and H2 cells compared with shCtrl cells 72 h post-induction. The results are presented as mean +/− standard deviation of three independent experiments (*p < 0.05; *** p < 0,0001). C: shCtr and SEMA6A-depleted A3 and H2 cells were plated on poly-l lysine coated slides and stained with Phalloidin (red signal). GFP reporter gene expression revealed successful silencing induction. The cells were counterstained with Hoechst to highlight nuclei. Scale bar 10 μm. D: WB analysis of activated RhoA (RhoA-GTP) pulled down from cell lysates and total RhoA on cell extracts from shCtrl and SEMA6A-depleted 2/59 cells, treated or not with 1 U/mL RhoA activator. The anti-tubulin antibody was used to validate equivalent amount of loaded proteins in each lane. E: Densitometric analysis of RhoA-GTP normalized to RhoA obtained from shCtrl and SEMA6A-depleted A3 and H2 cell populations treated or not with 1 U/mL RhoA activator. The results are presented as mean+/− standard deviation of three independent experiments (*p < 0.05). F: Cytoplasmic and nuclear fractions extracted from shCtr and A3 and H2 cells, treated or not with RhoA activator, were analyzed by WB for the expression of SEMA6A, phosphorylated and total YAP. Lamin A and α-tubulin were used to validate purity of nuclear and cytoplasmic extracts respectively. G: Densitometric analysis of p-YAP normalized to total YAP in cytoplasmic fraction of shCtrl, A3 and H2 cells treated or not with 1 U/mL RhoA activator. The results are presented as mean+/− standard deviation of three independent experiments (*p < 0.05; ** p < 0,001).. H: shCtr and SEMA6A-depleted H2 cells plated on poly-l lysine coated slides were treated or not with RhoA activator and stained with anti-YAP (red signal) or I: with Phalloidin (red signal). Scale bar 10 μm. L: the number of stress fibers containing cells from the experiment shown in panel I is reported as percentage and at least 200 cells were counted per experiment. The results are presented as mean+/− standard deviation of three independent experiments (*** p < 0,0001)