Figure 5

Effect of HIF-1α silencing on the hypoxia-induced protection against the etoposide-induced apoptosis. A, cells were transfected with 5, 20 or 50 nM HIF-1α siRNA, 50 nM non-targeting siRNA or with the transfection reagent alone (DMF) for 24 hours. Cells were then incubated under normoxia or hypoxia for 6 hours and total cell extracts were analyzed by western blot for HIF-1α protein level. A, B, C, cells were transfected with 50 nM HIF-1α siRNA or non-targeting siRNA for 24 hours. They were then incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e, 50 μM) for 16 hours. B, after the incubation, total RNA was extracted, submitted to reverse transcription and then to amplification in the presence of SYBR Green and specific primers. α-tubulin was used as the house keeping gene for data normalization. Data are given in fold-induction. C, caspase 3 activity was assayed. Results are expressed as means ± 1 SD (n = 3). *** p < 0.001 vs. normoxia ; §§§ p < 0.001 vs. normoxia+etoposide ; ΔΔΔ p < 0.001 vs. hypoxia ; °°° p < 0.001 vs. hypoxia+etoposide. D, PARP-1 and cleaved 85 kDa fragment were detected in total cell extracts by western blotting, using a specific mouse anti-PARP-1 antibody.