Fig 1: SEMA6A is a prognostic indicator in BRAF-mut melanoma patients. A: SEMA6A Immunohistochemical assessment in BRAF-wt and BRAF-mut melanoma specimens of RECI1 cohort. Pearson’s Chi-square test revealed a significant association between SEMA6A expression and BRAF mutation (p < 0,0001). B: Representative sections from 2 BRAF-wt and 2 BRAF-mut melanoma patients, stained with anti-SEMA6A antibody. C: Progression-free Survival (PFS) and D: Overall Survival (OS) in patients with with high and low SEMA6A (BRAF-mut advanced melanoma). E: Relapse-free survival (RFS) in patients with high and low SEMA6A (BRAF-mut early melanoma) from the cohorts TCGA and DFCI
Fig 2: SEMA6A expression predicts low efficacy of dabrafenib+trametinib in BRAF-mut melanoma patients. A: Progression-free Survival (PFS) and B: Overall Survival (OS) in patients with high and low SEMA6A from the cohort RECI2
Fig 3: Dabrafenib- and dabrafenib+trametinib-induced SEMA6A-RhoA-YAP axis activation rescues actin cytoskeleton in BRAF-mut melanoma cells. A: BRAF-mut 2/59 and BRAF-wt ME4405 cells plated on poly-l lysine coated slides were treated or not with dabrafenib, trametinib and their combination for 48 h and 7 days, and stained with anti-YAP (green signal) and Phalloidin (red signal). The cells were counterstained with Hoechst to highlight nuclei. Scale bar 10 μm. B: Quantification of the subcellular localization of YAP from immunofluorescence of 2/59 and ME4405 cells untreated and treated with dabrafenib, trametinib and their combination for 48 h and 7 days. The results are reported as percentage of YAP expression in cytoplasmic + nuclear and nuclear fractions and at least 200 cells were counted per experiment. The results are presented as mean+/− standard deviation of three independent experiments (*p < 0.05; ** p < 0,001; *** p < 0,0001)
Fig 4: 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
Fig 5: 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)
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