Figure 5. GSK126 causes cell growth inhibition via de-repression of tumor suppressor genes.

Microarray analysis of the top 30 most differentially expressed genes following 48 hr of 7.5 μM GSK126 treatment. Two sensitive (apoptosis) EZH2^Y646 mutant cell lines were selected (IGR1 and MM386) along with a sensitive WT (KMJR138) and an insensitive cell line (MEL-RMU) performed in duplicate. A. The top candidates with known tumor suppressor gene functions were validated by RT-qPCR in IGR1 B. and MM386 C. mutant cells. Western blot of cell lines treated for 48 hr with 7.5 μM GSK126 showing the effect on NDRG1_pT346 protein D. ChIP-qPCR (n = 2) in MM386 cells showing enrichment of H3K27me3 upstream of the transcription start site (primers (A–C) but not downstream (primer D) of the ATF3 gene promotor. CCND2 was used as a positive control and CDC6 as a negative control E. Survival analysis (Cox proportional hazard model) of reverse phase protein array (RPPA) data from 31 TCGA melanoma patients divided into high (>0.2963) or low (<0.2963) NDRG1_pT346 protein expression F. Survival analysis (Kaplan-Meier) of microarray data from 34 stage III melanoma patients [55] divided into high (upper 75^th percentile) or low (lower 25^th percentile) ATF3 mRNA expression G. Mutants are indicated in red.