Fig. 5.

ERK1/2 pathway inhibitors prime melanoma cells for rapid apoptosis following MCL1i. a, b A375 cells were treated with DMSO vehicle-only (Control) or 2 μM vemurafenib (Vem) for 24 h as indicated. One micromolar AZD5991 was then added for the indicated times. Apoptosis was assessed by Annexin V flow cytometry (a), or cells fixed, stained with propidium iodide and cell cycle profile determined by flow cytometry (b). c, d WM266-4 (c) and SK-MEL-30 (d) cells were treated with DMSO vehicle-only (Control), 1 μM vemurafenib (Vem) or 1 μM selumetinib (Sel) for 24 h as indicated. One micromolar AZD5991 was then added for the indicated times and apoptosis assessed by Annexin V staining and flow cytometry. e A375 cells were treated with DMSO vehicle-only (Control) or 1 μM selumetinib (Sel) for 24 h. One micromolar AZD5991 was then added for the indicated times and relative caspase-3/-7 activity measured. f A375 cells were treated with DMSO vehicle-only (Control) or 1 μM selumetinib (Sel) for 24 h followed by combination with 1 μM AZD5991 for the indicated times. BAX activation was determined using anti-BAX antibody clone 6A7, an N-terminal antibody specific for an active conformation of BAX, and flow cytometry. A representative flow cytometer trace is shown (left) alongside pooled results (right). g HCT116, HCT116 BAK KO, HCT116 BAX KO and HCT116 BAK BAX DKO isogenic cell lines were lysed and western blotted with the indicated antibodies. h, i HCT116, HCT116 BAK KO, HCT116 BAX KO and HCT116 BAK BAX DKO isogenic cell lines were treated with DMSO control (C), 2 μM selumetinib (Sel), 1 μM AZD5991 or 2 μM selumetinib plus 1 μM AZD5991 (Sel + AZD5991) with or without 20 μM Q-VD-OPh (Sel + AZD5991 + QVD) for 48 h, and cell death assessed by Annexin V (h) or PI (i) staining and flow cytometry. Results (a–f, h, i) are mean ± SD of three or more independent experiments. P ≤ 0.0001 (****) and ns (not significant) as determined by two-way ANOVA and Tukey’s multiple comparisons test