Fig 2. Effects of EZH2 on cholesterol efflux and ABCA1 expression.

A, DiI-oxLDL (1 μg/ml) was added to THP-1/RAW264.7 macrophages infected with LV-mock or LV-EZH2 vectors. After incubation for 24 h, the uptake was analyzed by flow cytometry. The control values were set to 100%, and then uptake data were calculated as the percentage of ox-LDL uptake vs. the control. B, Cholesterol efflux assay was performed using liquid scintillation counting assays as described in Methods. Cholesterol efflux of RAW264.7 macrophages were preloaded with [³H] cholesterol-oxLDL. C, EZH2 down-regulated ABCA1 mRNA in THP-1/RAW264.7 macrophages and mouse peritoneal macrophages (MPM). ABCA1 mRNA was quantified in THP-1 and RAW264.7 infected with LV-mock or LV-EZH2 vectors for 24 h or in MPM harvested from the peritoneal cavity of apoE^−/− mice 3 d after intraperitoneal injection with 2 ml thioglycollate. D, EZH2 down-regulated ABCA1 protein in both THP-1 and RAW264.7 macrophages. E, EZH2 downregulated ABCA1 protein in MPM harvested from the peritoneal cavity of apoE^−/− mice 3 d after intraperitoneal injection with 2 ml thioglycollate. Representative immunoblots show the effect of EZH2 on ABCA1 expression. Mean ± S.D., one-way ANOVA, *: P<0.05 and **: P<0.01 vs. control. F, EZH2 down-regulated ABCA1 mRNA in aorta, heart, liver and kidney of apoE^−/− mice. RNA from the indicated organs was isolated from apoE^−/− mice injected with LV-mock (n = 5, solid bars) or LV- EZH2 (n = 5, open bars). Mean ± S.D., one-way ANOVA, *: P<0.05 vs. HF+mock group. G, EZH2 downregulated ABCA1 protein in aorta, heart, liver and kidney of apoE^−/− mice. Representative immunoblots show the effect of EZH2 on ABCA1 expression. Proteins from the indicated organs were isolated from apoE^−/− mice treated with LV-mock or LV-EZH2 (n = 5).