Fig. 1

The anti-proliferative effect of EZH2 knockout does not persist in an EZH2-dependent cell population. G-401 and RD cells were infected with a lentiviral vector encoding Cas9 and each of three sgRNAs targeting EZH2. a The western blots show that 6 and 9 days after infection, EZH2 protein levels are decreased in both the G-401 cells and RD cells compared with a control sgRNA targeting the hemoglobin 1 (HBE1) gene. H3K27me3 levels are also decreased, whereas H3 levels are unchanged. The right hand panel indicates that treatment of G-401 cells and RD cells with a small molecule inhibitor of EZH2 for 72 h also decreases H3K27me3 levels, but has no effect on the expression levels of EZH2 and H3 as expected [6]. b, c Proliferation rate of cells from panel a. All 3 sgRNAs targeting EZH2 resulted in reduced rates of proliferation in G-401 cells and had little effect on the proliferation rate of the RD cells. d Proliferation rate of cells from panel a treated with or without a small molecule inhibitor of EZH2. e Western blot data showing that at 15 and 20 days post-infection, EZH2 expression levels are at near control levels in the G-401 cells infected with one of three independent sgRNAs targeting EZH2. A recovery in the levels of H3K27me3 was also evident. At the same time points, EZH2 protein expression remained low in RD cells infected with the same sgRNAs and H3K27me3 levels remained undetectable. f, g Proliferation rate of cells from panel e. The decrease in proliferation rates induced by EZH2 knockout was no longer evident at 20 days post-infection in the G-401 cells, while proliferation rates remain unaltered in RD cells expressing one of three independent sgRNAs targeting EZH2. All data in this figure are representative of at least two independent experiments. Error bars show standard error mean for six technical replicates