Figure 1.

The function and expression of EZH1 and EZH2 in MRT

(A) The functions of EZH1 and EZH2 on cell proliferation in MRT cells. A204.1, TTC642, and G401.6TG cells were infected with retroviral shEZH1 and/or shEZH2 vectors, and transduced cells were selected with 0.5 mg/mL (A204.1) or 0.1 mg/mL (TTC642 and G401.6TG) of G418 for 7 days. The relative cell growth of the transduced cells was measured by WST-8 formazan dye (OD 450) for the indicated durations (n = 3, means ± SD). Differences were statistically evaluated by two-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance between control and each KD condition (shEZH1, shEZH2, and shEZH1/2) was observed after day 11 in all cell lines. shEZH1 versus control is p < 0.0001, shEZH2 versus control is p < 0.0001, and shEZH1/2 versus control is p < 0.0001 in all cell lines. Statistical significances between single KD and double KD on day 14 are as follows: shEZH1 versus shEZH1/2 is ∗∗∗p < 0.0001 in all cell lines. shEZH2 versus shEZH1/2 is ∗∗∗p < 0.0001 in A204.1 and G401.6TG cells, but p = 0.4519 in TTC642 cells. (B) Expression of EZH1, EZH2, and H3K27me3 proteins in cells single or double KD of EZH1 and EZH2. The expression level of each protein was determined by western blotting analysis. β-actin and H3 were used as the internal controls. The numbers below EZH1, EZH2, and H3K27me3 indicate each band density relative to control (taken as “1”). (C) Expression of EZH1 and EZH2 mRNAs in cells single or double KD. Relative expression level of each mRNA was evaluated by qRT-PCR (n = 3, means ± SD). The expression level of the control is indicated as “1.” ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus control. The KD efficacy was also confirmed by expression levels of proteins (B) and mRNAs (C).