A. RT-qPCR analysis of SPANX expression in melanoma patient-derived xenografts (PDX).
B. RT-qPCR and western blot analysis of SPANX expression in A375 cells transduced with empty vector (EV) or SPANX-targeting shRNA (n=3). C. A375 cells transduced with EV or SPANX-targeting shRNA were monitored for altered viability using a Cell Titer Glo luminescence-based kit (n=4). Statistical significance was assessed by two-way ANOVA with post hoc Tukey’s test, ***p-value < 0.001. D. Colony formation assay (left) and quantification (right) of A375 colony number following transduction with SPANX shRNA or EV control (n=3). E. Viability assay based on crystal violet staining of MM150922 cells transfected with control (ctrl) or SPANX-targeting siRNA (left) and quantification (right, upper) (n=3). Statistical significance was assessed by Student’s t-test, *p-value < 0.05. (right, lower) Western blot analysis of SPANX expression in MM150922 cells transfected with ctrl or SPANX-targeting si-RNA. F. Western blot analysis of A375 cells transduced with EV (pLVX) or vector expressing SPANXA (A) or SPANXC (C). SPANX expression was evaluated 4 days after induction with doxycycline. G. Sphere formation assay of A375 cells transduced as in (F) and pre-treated for 3 days with doxycycline. (left) Representative pictures of the obtained spheroids. Scale bar: 100μm. (right) Calculation of sphere area 8 days after cell seeding (n=3, 6 spheres per independent experiment). Error bars represent means ± SEM. Statistical significance was assessed by one-way ANOVA with post hoc Tukey’s test, **p-value < 0.01, ***p-value < 0.001 in panels B, D and G.