Figure 5

HIFs regulate the expression of Parkin. (A and B) Hep3B cells were cultured under normoxia or hypoxia (O₂ = 1%) for 12 h, and the protein expression of Parkin and Hsc70 (A) and the mRNA level of Parkin (B) were assessed by immunoblotting and RT-PCR, respectively. (C) Hep3B cells were transfected with siRNA-Control, siRNA-HIF-1α (HIF-1α K.D.), or siRNA-HIF-2α (HIF-2α K.D.) and incubated under hypoxia (O₂ = 1%, 12 h). The mRNA abundance of Parkin, HIF-1α, HIF-2α, and β-actin was measured by RT-PCR. (D) Wild-type hypoxia response elements (HREs) and their mutated constructs (HRE mut 1 and 2) within the Parkin promoter − 360/ + 100 (upper panel) were inserted into the pGL3 vector. These constructs were co-transfected with pRL-null into Hep3B cells and then cultured under normoxia or hypoxia (O₂ = 1%) or treated with CoCl₂ (100 µM, normoxia) for 12 h. Luciferase activity was then measured. (E) Genomic positions of Parkin regions that were selected for the ChIP assay (left panel). Hep3B cells were incubated under normoxia or hypoxia (O₂ = 1%) or treated with CoCl₂ (100 µM, normoxia) for 12 h, and the ChIP assay was performed with the anti-HIF-1α antibody or control rabbit IgG, with input chromatin as the positive control. After reverse cross-linking, DNA was amplified using the indicated primer sets (right panel). Data are the mean ± S.D. of triplicate experiments. N.S. not significant, *p < 0.05, **p < 0.01, ***p < 0.001 versus the indicated group, calculated with a one-sample t-test with Bonferroni’s correction (for figure A), a one-sample t-test (for figures B), or a multivariate one-way ANOVA with Tukey’s post-hoc test (for figures C and D).