Fig. 7. The mitochondrial inhibitors NaN₃ and CCCP inhibit RBM3 and CIRP induction by hypoxia but superinduce VEGF expression by hypoxia. HeLa cells were cultured in parallel in normoxia or 1% oxygen as indicated for 24 hours. Different concentrations of either NaN₃ or CCCP were added as indicated. (A) RNA was isolated and subjected to real-time RT-PCR analysis. RBM3, CIRP and VEGF RNA levels were normalized to B2M RNA levels and are expressed as fold change in the different experiment samples compared with the corresponding normoxic samples (mean±s.e.m., n=3). To control for cell viability, cells were harvested and incubated with annexin V and propidiun iodide. Cell viability is given as the percentage of cells negative for both annexin V and propidium iodide staining (mean±s.e.m., n=3). (B) Annexin V reactivity and propidium iodide uptake of control HeLa cells (left dot blot), HeLa cells exposed to 5 mM NaN₃ (center dot blot) or HeLa cells exposed to 20 µM CCCP (right dot blot) for 24 hours.