Figure 5. Absence of MITF marks intrinsically insensitive cells.

(a) Treatment-naïve BRAF mutant melanoma cells were grouped based on their MITF expression by immunoblotting, also for additional proteins as indicated. HSP90 served as a loading control. (b) BRAF mutant melanoma cells were plated at low density and treated with 5 μM PLX4720 for 6 days or left untreated and stained with crystal violet. (c) For a subset of the cells lines from b, MAPK pathway inhibition and apoptosis indicated by cleaved PARP (arrow) after 3 days of treatment with 5 μM PLX4720 was confirmed by immunoblotting. HSP90 served as a loading control. (d) Sensitivity of MITF^endo_hi and MITF^endo_lo treatment-naïve melanoma cell lines to the BRAFi PLX4720, to the ERKi SCH772984 and to the MEKi trametinib was determined in dose–response curves. Mean was calculated from three independent experiments, error bars indicate s.d. (e) MITF^endo_hi and MITF^endo_lo BRAF^V600E mutant melanoma cell lines were plated at low density and treated with either BRAFi (2 μM), MEKi trametinib (0.1 μM) or a combination. After 6 days, cells were stained with crystal violet. (f) An independent set of treatment-naïve BRAF^V600E mutant melanoma cell lines was grouped based on MITF expression, and resistance to the BRAFi (vemurafenib) and the ERKi SCH772984 was determined by dose–response curves. Cell lines with MITF amplification are marked with an asterix.