Reoxygenation of anoxic cells results in a decrease in ATF-4 protein level, and proteasome inhibition results in an increase in ATF-4 protein level. (A) MDA-MB 435 and LB-4 cells were incubated for 16 hours in normoxia, hypoxia, or anoxia. After 16 hours, anoxic cells (or hypoxic cells, as in the case of MDA-MB 435 cells) were then immediately placed in a normoxic incubator for 40 minutes. This reoxygenation (reox.) of anoxic MDA-MB 435 and LB-4 cells resulted in the down-regulation of ATF-4 protein levels. Reoxygenation of hypoxic MDA-MB 435 cells did not affect the level of ATF-4 protein compared with hypoxic cells. (B) MDA-MB 435 cells were incubated for 16 hours in anoxia, after which the anoxic media of the cells were replaced with normal fresh media (oxygenated), and the cells were incubated in a normoxic incubator for 5 minutes. Cells were then analyzed by immunoblot for ATF-4 protein level. (C) MDA-MB 435 cells were incubated in normoxia for 16 hours, after which 200 or 500 μM (end concentration) of the proteasome inhibitor MG132 was added directly to the cells. Cells were incubated another 16 hours in normoxia or hypoxia and then analyzed using immunoblot for ATF-4 protein levels. N indicates normoxia; H, hypoxia; A, anoxia.