Effect of SCH-722984 on BRAF-mutant melanoma cell lines. A. IC50 (nM). 21 BRAF-mutant melanoma cell lines were exposed to 0–10 μM SCH-722984 (black bars) or vemurafenib (grey bars) and cell viability determined by ATP-based bioluminescence assay. Results are the mean of three experiments, performed in duplicates (n = 6). Error bars are standard deviation. Non-V600E substitutions are denoted in the bar graph for each corresponding cell line (M420, BRAFL597S; M381, BRAFV600R , M417, BRAFG466E, M414, BRAFV600K). Bar at 1 μM denotes threshold between sensitive and intermediate. Resistant cell lines have IC50 higher than 2 μM. B. Timecourse effects of SCH722984 on the MAPK signaling. SCH722984-sensitive M238, SCH722984-resistant M233, were treated in a timecourse manner with 500nM SCH722984 at 1, 2, 6, 12, 24 and 48 hours compared to DMSO as solvent control (C). Phosphorylated or total MEK, ERK1/2, RSK, AKT, or beta-actin as loading control were determined by western blot analysis. C. Effects of SCH722984 on the MAPK signaling at 24 hours. SCH722984-sensitive M262, SCH722984-resistant M381, SCH722984-intermediately sensitive M409 cells were treated for 24 h with DMSO as solvent control (-) or 500 nM SCH722984 (+).