Hypoxia protects HepG2 cells against etoposide-induced apoptosis. HepG2 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without etoposide (e) at 50 μM for 16 hours (A, B, C, D, F) or 40 hours (E). A, the overall caspase activity was assayed by measuring free rhodamine 110 released after cleavage of the caspase substrate DEVD-R110. Results are expressed as means ± 1 SD (n = 3). B, after the incubation, cells were fixed, permeabilized and stained for active caspase 3 using a specific antibody (green). Nuclei were detected with To-Pro-3 (blue). Observation was performed using a confocal microscope with a constant photomultiplier. C, after the incubation, DNA fragmentation was assayed using an ELISA for soluble nucleosomes. Results are expressed as means ± 1 SD (n = 3). 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. E, after the incubation, LDH was assessed. Results are expressed as means ± 1 SD (n = 3). F, after the incubation, complete medium was added back and cells were incubated for 7 days before being fixed and stained with crystal violet. **, ***: p < 0.01, p < 0.001 vs. normoxia ; §§§: p < 0.001 vs. normoxia+etoposide.