Hypoxia as well as proteasome and PHD inhibition stabilize ATF-4. (A) HeLa cells were treated for 8 hours with 300 nM thapsigargin or 10 μM MG132. Subsequently, cells were lysed and analyzed by immunoblotting. (B) HeLa cells were transiently transfected with an ATF-dependent firefly (FL) luciferase reporter gene plasmid (pATFx2-Luc) together with the renilla luciferase (RL) control plasmid pRLSV40. Following exposure to 20% or 1% O2, with or without the addition of 1 mM DMOG, cells were lysed and luciferase activities were determined. (C) TS-20 cells (with a temperature-sensitive E1 ubiquitin–activating enzyme defect) and E1-reconstituted H38–5 cells were exposed to 34°C or 39°C. Subsequently, cells were lysed and analyzed by immunoblotting. Shown are mean values of the FL/RL ratios (± SD) of 3 independent experiments.
