ATF-4 protein is induced in cancer cells under anoxia. (A) ATF-4 overexpression in normoxia (designated as +ATF-4) in MCF-7 cells confirmed the up-regulated band in anoxia to be ATF-4 (50 kDa). (B) Various cancer cell lines, including T47D, MDA-MB 231, 435, 468, and LB-4, were incubated for 16 hours under the corresponding oxygen levels and were analyzed by immunoblot for ATF-4 protein level. N indicates normoxia (21% O₂); A, anoxia. (C) MDA-MB 435 and murine N2a cells were incubated for 16 hours in either normoxia or anoxia and were analyzed by immunoblot for ATF-4 protein level in cytoplasmic and nuclear extracts. As indicated, ATF-4 induction in anoxia was only observed in nuclear extracts but not in cytoplasmic extracts. Nuc indicates nuclear; Cyt, cytoplasmic.