Fig. 5

Stress-induced JNK/JUN signaling is crucial for apoptosis induction by the combination of trametinib with CuET. A BRAF WT melanoma cells (SKMEL23 and SKMEL113) were treated with the MEK inhibitor trametinib (10 nM), CuET (125 nM), or their combination in the presence or absence (+/-) of the JNK inhibitor SP600125 (10 μM) for 12 hours. Whole cell lysates were used for Western blot analysis of phosphorylated JNK, total JNK, and JUN. β-Actin was used as a loading control. The results shown (cropped) are representative of two independent experiments. B Cell viability (MUH assays) of BRAF WT melanoma cells (SKMEL23 and SKMEL113) after treatment for 72 hours with trametinib (10 nM) plus increasing doses of CuET (up to 500 nM) in the presence or absence (+/-) of the JNK inhibitor SP600125 (10 μM). (n = 3, mean ± SD). The dotted line indicates the effect of 10 nM trametinib on cellular viability. C Flow cytometric cell cycle analyses of BRAF WT melanoma cells (SKMEL23 and SKMEL113) after treatment with MEK inhibitor trametinib (10 nM), CuET (125 nM) and trametinib plus CuET in the presence or absence (+/-) of the JNK inhibitor SP600125 (10 μM) for 24, 48 and 72 hours. Untreated melanoma cells were used as a control. The relative distribution of the cell cycle phases was analyzed (n = 3 independent experiments, mean ± SD)