Effect of tunicamycin, potassium cyanide, glucose deprivation, DFO, and CoCl2 on ATF-4 induction. (A) MDA-MB 435 cells were incubated for approximately 16 hours in normoxia after, which 200 μM DFO or CoCl2 (end concentration) was added directly to the cells. Cells were then analyzed after 16 hours by immunoblot for the indicated proteins: HIF-1α, ATF-4, GADD153, and β-tubulin (as an internal control). (B) MDA-MB 435 cells were incubated for 16 hours in normoxia and then stressed by either anoxia (A), tunicamycin (Tu; 1 mg/mL), arsenite (As; 2.5 μM), or cyanide (CN; 5 mM) for another 16 hours. Cells were analyzed by immunoblot for the indicated proteins. (C) MDA-MB435 cells incubated in glucose-free medium for 16 hours (–G) or in full medium and anoxia for 16 hours. Cells were then analyzed by immunoblot for the indicated proteins.
