Fig. 5

SEMA6A depletion restores responsiveness to dabrafenib and dabrafenib+trametinib in fibroblasts co-cultured BRAF-mut melanoma cells. A: Growth curves of mono-cultured and fibroblasts-cocultured shCtrl and SEMA6A-depleted A3 and H2 2/59 cells, periodically monitored up to 156 h. The results are presented as mean+/− standard deviation of three independent experiments (*p < 0.05). B: Fold change growth of co-cultured vs mono-cultured shCtrl and SEMA6A-depleted A3 and H2 cells as reported in A. C: Western blot (WB) analysis of SEMA6A, phosphorylated and total YAP, AKT, P65 and ERK was performed on total cell extracts from shCtrl and SEMA6A-depleted H2 cells cultured in the absence or presence of fibroblasts for 48 h. The anti-GAPDH antibody was used to validate equivalent amount of loaded proteins in each lane. D and F: Growth curves of mono-cultured and co-cultured shCtrl (D) and SEMA6A-depleted H2 cells (F), untreated or treated with 0,1 μM dabrafenib and 0,1 μM dabrafenib+ 5 nM trametinib, periodically monitored up to 156 h. E and G: the number of viable mono-cultured and co-cultured shCtrl (E) and SEMA6A-depleted H2 cells (G) 156 h post-treatment is reported. The results are presented as mean+/− standard deviation of three independent experiments (*p < 0.05; *** p < 0,0001). H: Growth curves of fibroblasts-cocultured shCtrl and SEMA6A-depleted A3 and H2 cells, untreated or treated as indicated, periodically monitored up to 180 h. The results are reported as Fold Change number of treated/untreated viable cells. I: Fold Change number of treated/untreated viable co-cultured shCtrl and SEMA6A-depleted A3 and H2 cells 180 h post-treatment is reported. The results are presented as mean+/− standard deviation of three independent experiments (*p < 0.05; **p < 0,001). L: WB analysis of SEMA6A, phosphorylated and total YAP, AKT, and ERK was performed on total cell extracts from mono-cultured and fibroblasts-cocultured shCtrl and SEMA6A-depleted H2 cells, untreated and treated as indicated for 48 h. The anti-HSP70 antibody was used to validate equivalent amount of loaded proteins in each lane