Figure 3

VBP1 negatively regulates HIF-1α but not HIF-2α.A, Western blot shows protein levels of HIF-1α but not HIF-2α were elevated in VBP1 knockdown HCT116 cells. The cells were stably transfected with the lentiviral pLKO vector containing specific shRNAs targeting VBP1 or control scrambled shRNA and exposed to either 21% or 1% O₂ for 6 h. B, HIF-1α protein levels were downregulated under hypoxic conditions. Western blotting analysis of control and VBP1-overexpressing HCT116 cells cultured at the indicated oxygen levels for 24 h. C–E, Overexpression of VBP1 decreased the HIF-1α but not HIF-2α protein levels. HCT116, U2OS, and HEK293T/17 cells were transfected with the indicated plasmids, and the proteins were detected by Western blotting analysis. (−), the cells transfected with the empty vector control; (+), the cells transfected with the indicated vector. F, Overexpression of VHL decreased HIF-2α protein levels but VBP1 failed to decrease it. 786-O cells were transfected with the indicated plasmids, and the proteins were detected by Western blotting analysis. G, ENO1 promotor-luciferase reporter activity in the HEK293T/17 and the HeLa cells show that VBP1 decreases HIF-1α but not HIF-2α transcriptional activity. The firefly luciferase activities were normalized to the Renilla luciferase activity. n = 3 biological replicate samples. H, VEGF promotor-luciferase reporter activity in the HEK293T/17 and the 786-O cells show that VBP1 failed to decrease HIF-2α or HIF-2α (P405A/P531A) transcriptional activity. The firefly luciferase activities were normalized to the Renilla luciferase activity. n = 3 biological replicate samples. I, RT-qPCR analysis of HIF target gene induction in response to hypoxia in HEK293T/17 cells transfected with empty vector or Flag-tagged VBP1 vector. The data are represented as means ± SD. n = 3 biologically independent extracts. ns, not significant. HIF-1, Hypoxia-inducible factor-1; pVHL, von Hippel-Lindau protein; VBP1, pVHL binding protein 1.