(a) and (b). Melanocytes stably expressing NRASG12D and GAB2WT+NRASG12D were plated onto 3D collagen gels for 24 hours in serum-free medium and analyzed for Annexin-V staining and PI uptake. Experiments were performed in triplicates. Error bars indicate ± standard error. *p<0.05.
(c) MM415 and MM485 are NRAS mutant whereas A2058 and G361 are BRAF mutant melanoma cell lines that overexpress GAB2. These melanoma cells were transfected with control or GAB2 siRNA. Forty-eight hours later cell lysates were analyzed for MCL-1, BCL-2, BCL-XL by Western blotting. Tubulin was used as loading control.
(d) and (e). Forty-eight hours following transfection with control and siGAB2, MM415, MM485, A2058, and G361 cells were replated onto 3D collagen for 48 hours in serum-free medium. Harvested cells were analyzed for Annexin-V staining and PI uptake. Bar graphs indicate percentage of Annexin-V positive cells. Representative traces from one of three experiments are shown. *p<0.05, **p<0.01, and ***p<0.001.