Figure 2. Methylation of the SYP promoter regulates the expression of SYP and cortisol secretion and H295R cells proliferation. (A) SYP methylation levels in CPA and normal adrenal tissues was determined using EpiTect Methyl II PCR Assay (n=12). (B) H295R cells were treated with 5-azaC, SYP methylation levels was determined by EpiTect Methyl II PCR Assay. (C) H295R cells were treated with 5-aza (5 μm) for 48 h the SYP protein level was determined by western blotting. (D) H295R cells were treated with 5-aza (5 μm) for 48 h, then cortisol secretion in H295R cell supernatants were measured in cell supernatants by ELISA. (E and F) Knock-down of SYP by siRNA was confirmed by qRT-PCR (F) and Western blotting (E). (G) The H295R cells was treated with SYP siRNA or control, then cortisol secretion in H295R cell supernatants were measured by ELISA. (H) Knock-down of SYP inhibited H295R cell proliferation measured by CCK assay. Three independent experiments were performed, and representative data are shown. Data are shown as mean ± SD. **p<0.01, compared with normal control. *p<0.05, compared with DMSO control.