Fig. 5

Parkin was a transcriptional target of FOXO3a. A, The promoter region of Parkin contains an optimal FOXO3a binding site. B and C, Immunoblotting results showing the protein levels of FOXO3a in cardiomyocytes infected with FOXO3a adenovirus or FOXO3a siRNA. n = 3 experiments per group. D and E, FOXO3a promoted PARKIN expression in cardiomyocytes. PARKIN increased upon overexpression of FOXO3a (D), while PARKIN decreased upon knockdown of FOXO3a (E). n = 3 experiments per group. F, Parkin promoter fragment containing FOXO3a potential binding site and its mutated fragment were cloned into luciferase reporter gene vector (pGL4.17), respectively (top). HEK293 cells were transfected with pGL4.17 vector expressing Parkin wide-type promoter or mutated promoter. The firefly luciferase activities were measured. ** p < 0.01 (bottom). n = 3 experiments per group. G, The firefly luciferase activities were detected in cardiomyocytes transfected with pGL4.17 vector expressing Parkin WT promoter and FOXO3a siRNA. ** p < 0.01. n = 3 experiments per group. H and I, Binding between FOXO3a and Parkin promoter was destroyed by Ang II. H Cardiomyocytes were exposed to Ang II, after transfected with pGL4.17 expressing Parkin WT promoter. The luciferase activities were attenuated upon Ang II treatment in a time-dependent manner. * p < 0.05. I Cardiomyocytes were treated with Ang II at the indicated time for ChIP analysis. Chromatin-bound DNA was immunoprecipitated with anti-FOXO3a antibody. Immunoprecipitated DNA was analyzed by PCR using a pair of primers combination that encompassed FOXO3a binding site. *** p < 0.001. **** p < 0.0001. n = 3 experiments per group