FIG. 4.

Blockade of autophagy in hypoxia triggers cell death through BNIP3. (A) Measurement of cell death in CCL39 cells. CCL39 cells were subjected to either normoxia (N) or hypoxia (H 1%) for 48 h before measuring cell death. (B) Measurement of cell death in DLD1 cells. Inset 1 shows the expression of BNIP3 and BNIP3L in DLD1 cells. The immunoblot shows DLD1 cells subjected to either normoxia (N) or hypoxia (H 1%) for 24 h. BNIP3 and BNIP3L expression was analyzed by immunoblotting. For the measurement of cell death, DLD1 cells were subjected to either normoxia (21% O₂) or hypoxia (1 and 0.1% O₂) for 48 h before measuring cell death. (C) Ablation of Beclin1 and ATG5 increase cell death in hypoxia. PC3 cells were transfected twice during a 24-h interval with siRNA to the control SIMA (40 nmol/liter), Beclin1 (40 nmol/liter), or ATG5 (40 nmol/liter), and cells were incubated for 48 h in hypoxia (H 1%) or normoxia (N). Beclin1 and ATG5 expression was analyzed by immunoblotting, and autophagy was examined. Two independent sets of siRNA to Beclin1 were used, but only one set of siRNA to ATG5 was used. Experiments were done at least twice. (D) Measurement of cell death in CCL39 clones overexpressing BNIP3/BNIP3L. CCL39 cells were incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before measuring cell death. Inset 2 shows the overexpression of BNIP3 in CCL39 cells. The immunoblot shows the overexpression of the BNIP3 protein in the presence of Tet. BNIP3 expression was analyzed by immunoblotting 3 days after Tet addition. The clone strongly expressed BNIP3 protein levels in normoxia. For the measurement of cell death, CCL39-pBNIP3 and CCL39-pBNIP3-BNIP3L clones were incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before measuring cell death. Inset 3 shows the overexpression of BNIP3 and BNIP3L in CCL39 cells. The immunoblot shows the stable overexpression of BNIP3L and the overexpression of the BNIP3 protein in the presence of Tet. BNIP3 expression was analyzed by immunoblotting 3 days after Tet addition. The clone strongly expressed BNIP3 and BNIP3L protein levels under normoxia. For cell death measurement, CCL39-pBNIP3-BNIP3L clones were incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before measuring cell death. (E) Measurement of cell death in Tet-induced BNIP3-expressing DLD1 cells. Inset 4 shows the expression of BNIP3 and BNIP3L in the DLD1 pBNIP3 clone. The immunoblot shows the DLD1 pBNIP3 clone incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before incubation for 24 h in hypoxia (1% O₂). BNIP3 and BNIP3L expression was analyzed by immunoblotting. For the measurement of cell death, the DLD1 pBNIP3 clone was incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before incubation for 24 h in hypoxia (1% O₂) before measuring cell death. (F) Ablation of BNIP3 increases cell death in normoxia and hypoxia. Inset 5 shows the effect of BNIP3 ablation. The immunoblot shows the ablation of BNIP3 in normoxia and hypoxia. LS pTerBNIP3 cells were incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before incubation for 48 h in hypoxia (H 1% O₂). BNIP3 and BNIP3L expression was analyzed by immunoblotting. Hsp90 was used as a control. For the measurement of cell death, the LS pTerBNIP3 clone was incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before incubation in either normoxia (N) or hypoxia (H 1%) for 48 h. Inset 6 shows the effect of BNIP3 and BNIP3L ablation. The immunoblot shows the ablation of BNIP3L in normoxia and hypoxia in the absence of BNIP3 expression. The LS pTerBNIP3 clone was incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before transient transfection with siRNA to BNIP3L (40 nM). Cells then were subjected to normoxia (N) or hypoxia (H 1%) for 48 h. BNIP3 and BNIP3L expression was analyzed by immunoblotting. Hsp90 was used as a control. For the measurement of cell death, the LS pTerBNIP3 clone was incubated in the absence (−Tet) or presence (+Tet) of Tet (10 μg/ml) for 3 days before transient transfection with siRNA to BNIP3L (40 nM). An siRNA to SIMA (siSIMA; 40 nM) was used as a control. Cells then were subjected to normoxia (N) or hypoxia (H 1%) for 48 h before measuring cell death with trypan blue. Experiments were done in duplicate at least three times.