EZH2 overexpression modulates HPASMC phenotype.

<p>The HPASMCs were transfected with EZH2 or control plasmid and expression levels of calponin, a SMC phenotypic marker was assessed for A) mRNA by real-time PCR and B) protein levels by Western blot. All experiments were repeated at least 3 times and graph plotted is an average of three independent experiments. P<0.005 compared to controls. C) EZh2 increases binding to calponin-1 promoter in HPASMCs. DNA fragments interacting with H3K27me3 were pulled down by anti H3K27me3 or normal rabbit antibody. The presence of Calponin promoter DNA was checked by PCR using calponin promoter specific primers (upper panel). MYOD1 promoter segment binding to H3K27me3 was used as a positive control (lower panel). Calponin promoter region was amplified in EZH2 transfected cells and immunoprecipitated with anti-H3K27me3 but not in vector transfected cells (lanes 2 and 5). The immnoprecipitation with normal rabbit IgG did not yield any PCR amplification (lanes 3 and 6).