Figure 4: Hypoxia response elements (HREs) in the H19 promoter are not the only mechanism to mediate Hif-1α-induced H19 expression.

(A) Sequences and locations of the three HREs within 2 kb before the transcription start site in the H19 promoter. The wild-type HRE consensus sequence of designed luciferase report construct was 5′-ACGTG-3′, and the mutant HRE sequence was 5′-AATAT-3′. (B) Chromatin immunoprecipitation assays were performed to indicate binding of Hif-1α to the HREs located in the H19 and VEGF promoters. Mouse anti-Hif-1α antibody or control mouse IgG was used for immunoprecipitation with DNA isolated from U87 and U251 cells. The immunoprecipitate was amplified by qPCR using primers targeting the HREs. The results were normalized to the negative control IgG. The HRE in the VEGF promoter was used as positive control. (C) Five luciferase reporter plasmids were designed containing different combinations of wild type and mutated HREs (schematized on the left). Relative luciferase activity was observed for each reporter construct following transfection of empty vector or Hif-1α (horizontal bars, right) in U87 and U251 cells with 24 h hypoxia treatment. Renilla activity was used as an internal loading control. (D) The relative luciferase activity for each reporter construct following transfection with the same Hif-1α plasmid normalized to the WT group. (*p < 0.05, **p < 0.01).