Fig. 4

MITF directly targets IDH1 and NNT, and they protect melanoma from oxidative damage. Similar to the microarray dataset, we found significant correlations of MITF with NNT (A) and IDH1 (B) across 54 melanoma cell lines in the CCLE RNA-sequencing datasets and in the (n = 472), (n = 287) (C, D) TCGA RNA-sequencing datasets. (Total available melanoma cell lines for RNA-seq data at the Depmap portal: 54) NNT mRNA levels following knockdown (E) or overexpression (F) of MITF (48 h) in human melanoma cells. IDH1 mRNA levels following knockdown (G) or overexpression (H) of MITF (48 h) in human melanoma cells. ChIP with polyclonal rabbit anti-MITF antibody in UACC257 melanoma cells. Precipitated DNA was amplified using primers surrounding a MITF binding site within the NNT (I) or IDH1 (J) promoter region and compared to rabbit IgG control. Effects of MITF overexpression on luciferase reporter activity driven by the NNT and IDH1 promoter (K, L) with wild type (WT) or impaired (MUT) E-BOX sequences in UACC257 cells. Representative flow plots for cytosolic ROS levels measured by DCFDA fluorescent dye after knockdown of MITF, IDH1, NNT, or PGC1α in UACC257 melanoma cells (M) and in SKMEL30 melanoma cells (N). MITF, IDH1, or NNT silencing induced ROS-dependent melanoma death that is rescuable by 2 mM NAC (N-Acetyl-L-Cystein) in UACC257 melanoma cells (O). Values represent the SD of three independent experiments performed. P values in m and n indicate significance compared to siControl. Non-targeting siRNA (siControl) was used as negative control. All mRNA levels were normalized to RPL11. All luciferase activity were normalized to Renilla. Values represent the mean ± SEM. *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001. Abbreviations: MITF: Microphthalmia-associated transcription factor, NNT: Nicotinamide nucleotide transhydrogenase, IDH1: Isocitrate dehydrogenase 1, CCLE cancer cell line encyclopedia, TCGA The Cancer Genome Atlas, TYR tyrosinase, ACTB Actin-beta, PGC1a peroxisome proliferator- activated receptor gamma coactivator-1 alpha, NAC N-acetyl-L-cystein.