Effect of hypoxia on paclitaxel or epirubicin-induced apoptosis and cell death. MDA-MB-231 cells were incubated under normoxic (N) or hypoxic (H) conditions with or without paclitaxel (tax, 50 μM) or epirubicin (epi, 10 μM) for 16 hours. (A) Caspase 3 activity was assayed by measuring free AFC released from the cleavage of the caspase 3 specific substrate Ac-DEVD-AFC. Results are expressed in fluorescence intensity, as mean ± 1 SD (n = 3). (B) After the incubation, LDH release was assessed. Results are expressed as mean ± 1 SD (n = 3). (C) After the incubation, DNA fragmentation was assayed using an ELISA for soluble nucleosomes (Cell Death Detection Elisa, Roche). Results are expressed as mean ± 1 SD (n = 3). (D) Nuclear fragmentation was observed after nuclei labeling with DAPI using non-confocal fluorescent microscope (40x magnification). Fragmented nuclei are pointed by arrows. N.S. = non significantly different from control, ** = significantly different from control (p < 0.01), *** = significantly different from control (p < 0.001) ; N.S. = no significant difference between N epi and H epi, ## = significant difference between N tax and H tax (p < 0.01). ### = significant difference between N tax and H tax (p < 0.001).