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. 2013 Mar 1;288(15):10703–10714. doi: 10.1074/jbc.M112.414771

FIGURE 1.

FIGURE 1.

CMA inhibitors increase HIF-1α protein levels. A, HeLa cells were left untreated or treated with bafilomycin (10 nm) for 24 h, after which RNA was isolated, and RT-qPCR of the indicated HIF target genes was performed. B, HeLa cells were transfected with the HIF-1 reporter plasmid p2.1 and the Renilla luciferase reporter pSV-RL, exposed to 1% O2, and left untreated or treated with bafilomycin (10 nm) for 48 h. Afterward, cells were lysed, and reporter activity was measured. C, Hep3B cells were transfected with FLAG-HIF-1α expression vector that was either unmutated (WT) or harbored P402A/P564A mutations (double mutant (DM)). 24 h post-transfection, the cells were left untreated or treated with bafilomycin (Baf; 10 nm) or chloroquine (CQ; 50 μm) for an additional 8 h, after which cells were lysed, and lysates were probed with the indicated antibodies. WB, Western blot. D, Hep3B cells were left untreated or treated with the proteasomal inhibitor MG132 (10 μm) either alone or in combination with bafilomycin (10 nm) or chloroquine (50 μm) for 8 h. Afterward, cells were lysed, and lysates were probed with the indicated antibodies. Hep3B (E) and HeLa (F) cells were left untreated at 20% O2 (normoxia); treated with bafilomycin (10 nm), chloroquine (50 μm), or NH4Cl (10 mm); or left untreated and exposed to 1% O2 (hypoxia) for 8 h. Afterward, cells were lysed, and lysates were probed with the indicated antibodies. RNA was also isolated, and RT-qPCR of HIF-1α mRNA was performed. Normal human foreskin fibroblasts (NuFF; G) and MEFs (H) were left untreated at 20% O2; treated with bafilomycin (10 nm), chloroquine (50 μm), or NH4Cl (10 mm); or left untreated and exposed to 1% O2 (hypoxia (H)) for 8 h. Afterward, cells were lysed, and lysates were probed with the indicated antibodies. I, Hep3B cells were left untreated; treated with bafilomycin (10 nm), chloroquine (50 μm), or NH4Cl (10 mm); or exposed to 1% O2 for 24 h. Afterward, RNA was isolated, and RT-qPCR of the indicated HIF target genes was performed. J, Hep3B cells stably infected with empty shRNA vector or vectors against both HIF-1α and HIF-2α (shHIF-1α/2α) were left untreated or treated with bafilomycin (10 nm) or chloroquine (50 μm) for 24 h. Afterward, RNA was isolated, and RT-qPCR of the indicated HIF target genes was performed. K, Hep3B cells were treated with various combinations of bafilomycin (10 nm), chloroquine (50 μm), and the HIF-1 inhibitor acriflavine (ACF; 5 μm) for 24 h. Afterward, RNA was isolated, and RT-qPCR of the indicated HIF target genes was performed. Results are shown as means ± S.E. *, p < 0.01; #, p < 0.05.

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