FIGURE 2:

Bmi1 expression is directly regulated by HIF-1α. (A) qRT-PCR and Western blot analysis of Bmi1 mRNA and protein in siR-HIF-1α– and control vector–transfected HK-2 cells under low oxygen. (B) Relative luciferase activity of the Bmi1 promoter reporter gene. HK-2 cells were transfected with 20 ng of reporter constructs and 1 mg of pcDNA3.1-HIF-1a in combination with 0.2 ng of pRL-TK vector for 24 h. The luciferase results are reported as relative light units of firefly luciferase activity normalized with respect to the Renilla luciferase activity. Mean values from three independent experiments. *p < 0.01 compared with control. (C) Chromatin immunoprecipitation was performed to examine HIF-1a binding to the Bmi1 promoter in HK-2 cells under normoxia and low oxygen. Reaction controls include immunoprecipitations performed by using a nonspecific IgG monoclonal antibody (Cont IP), and PCR was performed by using whole-cell genomic DNA (Input). Representative example of three independent experiments.