FIG. 5.
Hypoxia triggers autophagy in promoting survival. (A) Coimmunoprecipitation of endogenous Beclin1 with Bcl-2. PC3 cells were subjected to hypoxia (H 1%; 48 h), and cell lysates were immunoprecipitated with an anti-Beclin1 antibody resin. The presence of Beclin1 protein in the lysates and the immunoprecipitates is shown (arrow). IB, immunoblot; IP, immunoprecipitate. (B) Sequences of the three BH3 peptides used, BNIP3L, BNIP3, and a mutated form of BNIP3 (BNIP3 mut). The stretch of arginine residues (R8) and the glycine linker (G) are shown in italics, and the central core of the BH3 domain is in boldface. B3, R8 peptide of the BNIP3 BH3 domain; B3L, R8 peptide of the BNIP3L BH3 domain; AA, R8 peptide of the mutated BNIP3 BH3 domain. (C) R8-BH3 peptides of BNIP3 and BNIP3L trigger autophagy (i.e., an increase in the LC3-II/LC3-I ratio) in normoxia in LS174 cells. LS174 cells were exposed in complete medium three times during a 24-h period to 25 μg/ml of the R8-modified peptides in normoxia. Experiments were done twice in LS174 cells, giving identical results (results from one experiment are shown). In the two experiments, the addition of the mutated AA form gave results identical to those for dimethyl sulfoxide (DMSO) with no peptide. Total cell extracts were analyzed by immunoblotting with antibodies against LC3 and β-actin. (D) Disruption of the Bcl-2-Beclin1 complex by BH3 peptides derived from BNIP3 or BNIP3L. An acellular assay was performed with a normoxic lysate of PC3 cells incubated in the absence or presence of BNIP3 or BNIP3L peptide. The coimmunoprecipitation of endogenous Beclin1 was performed with an anti-Bcl-2 antibody. The presence of Beclin1 in the lysates and the immunoprecipitate is illustrated (arrow). (E) BNIP3 and BNIP3L peptides trigger autophagy in stable GFP-LC3-expressing PC3 cells. PC3 cells were infused five times with the AA (20 μM), BNIP3 (20 μM), or BNIP3L peptide (20 μM) for 24 h. An example of GFP-LC3 punctate structures observed in GFP-LC3 PC3 cells under normoxic and hypoxic conditions is given. GFP fluorescence was examined under a Zeiss fluorescence microscope. The quantification of the number of GFP aggregates per cell was performed using the analyze particles tool in the Image J software. *, P < 0.00001.