Figure 3
EZH2 inhibition upregulates cell cycle control genes to exert its antiproliferative effect. (a) Fold change in mRNA levels in EPZ005687- and UNC1999-treated KMS11 and KMM1 cell lines at 3 and 6 days, compared with DMSO control at the same time point, measured by qRT-PCR. Graphs show mean and s.e.m. for at least three independent replicate experiments. GAPDH was used as the internal control. (b) p15 and p21 immunoblotting of lysates from KMS11 and KMM1 cell lines after 3 and 6 days’ incubation with EPZ005687. Actin was used as the loading control. Blots shown are representative of two independent experiments. (c) ChIP with H3K27me3 antibody followed by qRT-PCR at regions indicated in samples incubated for 6 days with DMSO control or EPZ005687 at the indicated concentrations. Pulldown of genes is shown as the percentage of input. Active motif negative and positive controls were used (mapping to genes ACTB and MYT1, respectively). An additional region approximately 5 kb (see supplementary Methods) upstream of each gene of interest was also assayed. Isotype control antibody led to negligible pulldown (data not shown). (d) Kaplan–Meier curves showing OS in the UAMS-TT data set comparing patients with low CDKN1A expression (<10.6, log 2 expression value n=105) to all others (n=1125). Median OS 3.56 years (95% CI (2.87, 6.17)) vs 10.9 (95% CI (10.1, 12.4)). Logrank P=6.1e-12.
