Figure 2.
PDGFR family signaling promotes hypoxia-induced autophagy of cytosolic protein. (A) BE GFP-LC3 cells at confluence were cultured in serum-free medium (SFM) (21% O2) or EBSS (no amino acids [no a.a.]) or glucose-free medium (no glucose), all at 21% O2, or in SFM at 1% O2 (1% O2), plus DMSO vehicle or PDGFR-I (5 μM) for 8 h, whereupon cells were fixed and analyzed for GFP-LC3 puncta by fluorescence microscopy (n = 3 ± SEM; [*] P < 0.05; [**] P < 0.01; [***] P < 0.005 vs. DMSO control). (B) Parental BE cells (no GFP-LC3 transgene) were cultured in SFM under normoxia (21% O2) or hypoxia (1% O2) plus DMSO or PDGFR-I (5 μM) for 4 h and then lysed and immunoblotted for LC3 isoform quantification. (C) BE GFP-LC3 cells at confluence were cultured in SFM under normoxia (21% O2) or hypoxia (1% O2) plus DMSO or PDGFR-I (5 μM) for 4 h and then lysed and immunoprecipitated for PDGFRβ and probed for phospho-tyrosine and PDGFRβ. (D,E) BE GFP-LC3 cells were transfected with indicated siRNA oligonucleotides (nontargeting control [NTC1], or PDGFRβ or FMS targeting) and at 36 h post-transfection cultured in SFM under normoxia (21% O2) or hypoxia (1% O2) overnight before scoring of the number of GFP-LC3 puncta-positive cells (n = 3 ± SEM; [**] P < 0.01; [***] P < 0.005; [#] not significant vs. NTC1). Silencing of targets was confirmed by immunoblotting (D, inset) or qPCR (E) (numbers in parentheses indicate relative mRNA levels). (F,G) BE GFP-LC3 cells at confluence were cultured in SFM under normoxia (21% O2) or hypoxia (1% O2) plus DMSO, PDGFR-I (5 μM) (F) or E64d (10 μg/mL) plus pepstatin A (10 μg/mL) (G) for 8 h (F) or 24 h (G), and then lysed and immunoblotted for indicated proteins. (H) BE GFP-LC3 cells and primary MEFs at confluence were cultured in SFM under normoxia (21% O2) or hypoxia (1% O2) for indicated times and lysed and immunoblotted for indicated proteins. See Supplemental Figure 2J for quantification.