Figure 1.

(a) Apoptosis induction is shown in the melanoma cell line A-375 at 24 and 48 h of treatment with vemurafenib (Vem; 3, 10, 30 μM) and TRAM-34 (TRAM; 25, 50, 100 μM). Apoptotic effects with the concentrations of 30 μM Vem and 50 μM TRAM, used for most subsequent experiments, have been reproduced multiple times (>3 × ). Apoptosis values correspond to cells with a less DNA content than cells in G1 phase, which is due to DNA fragmentation (sub-G1). Examples are given on the right side. Cell populations in G1, G2 and S-phase as well as sub-G1 cells are indicated. (b) For three conditions (24 h, TRAM-24, 25 μM and 50 μM; 48 h, 50 μM TRAM-24), calculated additive effects on apoptosis (diamond symbols) are directly compared with experimentally determined combination effects (circle symbols, corresponding to panel (a)). Calculated additive effects result from direct addition of apoptosis by vemurafenib and apoptosis by TRAM-34. For further comparison, apoptosis by vemurafenib alone (3, 10, 30 μM) is shown (square symbols). (c) Melanoma cell lines Mel-HO and Mel-2a were treated with selected concentrations of Vem (30 μM) and TRAM (50 μM). (d) Combinations of TRAM (50 μM) and Vem (10, 30 μM) were used for treatment of an A-375 cell population selected for vemurafenib resistance. All experiments have been performed with triplicate values, and at least two independent experiments for panels (c and d) revealed highly comparable effects