Fig. 4

Induction of ER stress-related genes by trametinib plus CuET in BRAF WT melanoma cells. A Top 50 differentially expressed genes were clustered in a heatmap showing log10 (fold change expression) values between the monotherapies and the combination using untreated cells as a reference. SKMEL23 cells were treated with trametinib (10 nM), CuET (125 nM) or the combination for 12 hours in triplicate. Total RNA was isolated, and RNA sequencing (RNA-seq) was performed to calculate differentially expressed genes. B RNAseq data of SKMEL23 cells revealed the induction of ER stress-related genes due to the combination treatment with trametinib (10 nM) plus CuET (125 nM) for 12 hours (n = 3; mean ± SD). C Quantitative real-time qPCR using RNA isolated from BRAF WT melanoma cells (SKMEL23 and SKMEL113) treated with trametinib (10 nM), CuET (125 nM) or their combination for 12 hours. The mRNA expression of ATF4, CHOP and NUPR1 was normalized to that of TBP and ACTN (n=3, mean ± SD). The results were analyzed using one-way ANOVA with subsequent Tukey’s multiple comparisons test. D BRAF WT melanoma cells were treated with trametinib (10 nM), 125 CuET (125 nM) and the combination for 12 hours. The ER stress inducer HA15-1 (1 μM) served as a positive control. Western blots (cropped) for ATF4, p8/NUPR1, and CHOP were performed with whole cell lysates, and β-Actin was used as a loading control. A representative result of two independent experiments is shown