Figure 8: EZH2 target genes are functionally involved in melanomagenesis.

(a) Gene expression signature of M010817 and M050829 after EZH2 depletion using siE. Average log2 fold change values of three siE replicates compared with three siCo replicates in (Supplementary Fig. 10) and significantly changed in both cell lines are shown. (b,c) Summary of TCGA-based unbiased analyses of genes upregulated (b; Supplementary Table 2) or downregulated (c) in (a). For upregulated genes in (a), specimens with high RNAseq reads for the corresponding genes were compared with specimens with low RNAseq reads. For downregulated genes in (a), specimens with low RNAseq reads for the corresponding genes were compared with specimens with high RNAseq reads. (d) Reverse transcription–qPCR (RT–qPCR) for genes associated with improved survival in (b) on M010817 and M050829 after EZH2 depletion using siE (relative to GAPDH). (e) H3K27me3 ChIP and subsequent qPCR for promoters of genes associated with improved survival in (b) on untreated M010817 and M050829. (f) Quantification of G1/(S+G2M) ratio of M010817, M050829 and M990514 after depletion of EZH2 and DCK/WDR19 using siE and siDCK/siWDR19. (g) Quantification of relative invasive capacity of M010817, M050829 and M990514 after depletion of EZH2 and AMD1 using siE and siAMD1. (h) H3K27me3 ChIP and subsequent qPCR for gene promoters of selected ETGs on M010817 and M050829 after EZH2 depletion using siE. (i) RT–qPCR for selected ETGs on lysed shCo, shE and GSK503-treated B16-F10 tumours from (Supplementary Fig. 8b). (j) RT–qPCR for selected ETGs on lysed control, cKO and GSK503-treated RIM-1 tumours from (Fig. 6f). Data are represented as mean±s.e.m. of n=3 (d,f–h,j), mean±s.e.m. of n=4 (e), mean±s.e.m. of n=3 (shCo), n=4 (shE, GSK503) (i). P values calculated with analysis of variance and Fisher’s least significant difference test.