Fig. 3

Direct binding of HIF-2α to the APOL1 promoter. (A) Quantitative real-time PCR and Western blot detected the interference efficiency of APOL1 in indicated cell lines. (B) Representative images of HIF-2α and APOL1 expression in ccRCC serially cut tissue by IHC. (C) Correlation between the expression of HIF2α protein and APOL1 protein in clear cell renal cell carcinoma (n = 150). (D) The reads distributed on two sides of the transcription start site (TSS). (E) ChIP peaks over chromosomes by ChIP-seq technology employing the primary antibody against HIF-2α (GSM5573436). (F) ChIP-seq enrichment profiles of HIF-2α in genomic region spanning the VEGFA (positive control) and APOL1. (G) Diagram of the APOL1 promoter region analyzed for putative HREs (green boxes) from the − 2000 to the transcriptional start site of APOL1 (+ 1). Three putative HREs located at different sites in the APOL1 promoter sequence. Primer pairs used for PCR amplification after ChIP are indicated. Primer 1 pairs (P1) amplified product including HRE3, Primer 2 pairs (P2) amplified product including HRE2, Primer 5 pairs (P5) amplified product including HRE1. Results of ChIP-real-time PCR and ChIP-PCR assay conducted using chromatins isolated from 786-O cells. A specific anti- HIF-2α antibody was used, and normal IgG was used as a control. 2% of the total cell lysates were subjected to PCR before immunoprecipitation (input control). The experiments were performed three times independently. (H) The fragment including putative HRE were structured into pGL4.10 plasmid (pGL4.10-923-2 including putative HRE3, HRE2 and HRE1, pGL4.10-923-3 including putative HRE2 and HRE1, pGL4.10-923-4 including putative HRE1, pGL4.10-923-4-mu including the mutant HRE1). APOL1 promoter reporters (pGL4.10-923-2, 923-3, 923-4 and 923-4 mutant) and pGL4.73 were co-transfected into 239T cells for 24 h. The APOL1 promoter activity was then examined using a dual luciferase assay kit