Kinetics of hypoxic stabilization and reoxygenation-induced destabilization of ATF-4. (A) HeLa cells were incubated at 20%, 1%, or 0.2% O₂, with or without the addition of 1 mM DMOG. After 4 or 24 hours, the cells were lysed and analyzed by immunoblotting. As a control for the induction of ATF-4 by ER stress, one dish of HeLa cells was treated with 300 nM thapsigargin for 4 hours. (B) HeLa cells were incubated at 0.2% O₂ with or without the addition of 1 mM DMOG. At 4 hours later, 20 μg/mL cycloheximide was added, and cells were reoxygenated at 20% O₂. After the indicated time points, cells were lysed and analyzed by immunoblotting.