Figure 1

dnHIF expression reduces expression of HIF-1 target proteins (CA-9 and VEGF) and HIF-1α activity. A. Western blot analysis of HIF-1α, dnHIF and CA9 protein level in MDA-MB-231 wt, EV and dnHIF cells. Cells were exposed to normoxia (N) or hypoxia (H) for 18 h. β-actin detection served as the loading control. B. Fluorescent images of MDA-MB-231 EV and dnHIF cells stained with eGFP/dnHIF (green), CA9 (red) and DAPI (blue). Cells were exposed to normoxia or hypoxia for 18 h. C. HIF-reporter assay in MDA-MB-231 cells stably-expressing dnHIF or empty vector (EV) control following 18 h hypoxic exposure. Luciferase activity per μg protein was calculated and activity was normalised to EV control cells (**: p < 0.01 in dnHIF vs. EV cells). All results are from at least 3 independent experiments ± SD. D. VEGF levels in media taken from dnHIF and EV control cells in normoxia/hypoxia for 18 h. VEGF media levels were normalised to mg protein within the cell cultures from which the media were taken. Hypoxia significantly increased VEGF expression in control MDA-MB-231 EV cells (**: p < 0.01 in hypoxic vs. normoxic cells) but not in dnHIF cells. Most importantly, inhibition of HIF by use of a dominant-negative protein variant (dnHIF) inhibited hypoxia-induced VEGF expression (*: p < 0.05 in dnHIF vs. EV cells). All results are from at least 3 independent experiments ± SD.