Figure 3.

FoxO3A knockdown cells show increased sensitivity to hypoxia-induced apoptosis. (A, B) HeLa Ctrl and FoxO3A-KD#1 cell clones (A) and U2OS cells transiently transfected with siRNA oligos (B) were plated in triplicate and 24 h later incubated in normoxia or hypoxia (0.2% O₂). Every 24 h, samples were trypsinized, stained with trypan blue and viable cells were counted. Error bars indicate mean±s.d. of three independent experiments. (C, D) HeLa Ctrl and FoxO3A-KD#1 cell clones (C) and U2OS cells transiently transfected with siRNA oligos (D) were incubated for 24 h in normoxia or hypoxia (0.2% O₂) and subsequently analysed by immunoblotting. (E) HeLa Ctrl and FoxO3A-KD#1 cell clones were transfected with pBabe Puro or pBabe BCL-XL expression plasmid and subsequently incubated for 48 h in normoxia or hypoxia (0.2% O₂) in the presence or absence of Z-VAD. All cells were collected, stained with trypan blue, and live/dead cell numbers were determined. Error bars indicate mean±s.d. of three independent experiments, ^*P<0.05, ^**P<0.01 using Student's t-test. (F) HeLa Ctrl and FoxO3A-KD#1 cell clones were incubated for 48 h in normoxia or hypoxia (0.2% O₂) in the presence or absence of 100 μM antioxidant Trolox or solvent control. All cells were collected, stained with trypan blue, and live/dead cell numbers were determined. Error bars indicate mean±s.d. of three independent experiments, ^*P<0.05 using Student's t-test. (G) HeLa Ctrl and FoxO3A-KD#1 cell clones preincubated in the absence or presence of 100 μM antioxidant Trolox were incubated for 24 h in normoxia or hypoxia (0.2% O₂) and subsequently analysed by immunoblotting.