Figure 3.

(a) Protein expression was determined by Western blotting in A-375 cells at 24 and 48 h in response to treatment with 30 μM vemurafenib (Vem), 50 μM TRAM-34 (TRAM) and the combination (Combi). Expression was compared with non-treated cells (Ctr). Proteins analyzed: phosphorylated ERK (pERK), total ERK (ERK), antiapoptotic factors (Mcl-1, Bcl-2, XIAP), proapoptotic factors (Puma, Bim_EL, Bax, p53), and caspase-3 (proform, 35 kDa; active, cleaved forms, 15 and 17 kDa). Each determination was repeated with a second independent series of protein extracts, which showed highly comparable results. (b) The significance of caspase activation was proven by additional treatment with the pan-caspase inhibitor QVD-OPh (QVD, 10 μM). (c and d) Loss of MMP was determined by the potential-sensitive dye TMRM⁺ at 4 and 24 h of treatment in A-375 and Mel-2a. Examples, as determined by flow cytometry, are shown in the right panels. Cells with low MMP are indicated. (e) Apoptosis was determined in a Bcl-2-overexpressing A-375 cell clone (A375-Bcl-2). It was compared with a sensitive mock/plasmid-transfected A-375 cell clone (A375-pIRES). (b–d) All determinations were carried out in triplicates; asterisks indicate statistical significance