Induction of ATF-4 in anoxia does not require pVHL or HIF-1α. (A) Renal cancer cell line 786-0, deficient in pVHL (VA) or expressing pVHL (+pVHL), was incubated for 16 hours in normoxia or anoxia and analyzed after 16 hours by immunoblot analysis for ATF-4 protein level and HIF-2α. VA indicates vector alone (ie, not expressing pVHL): N, normoxia; A, anoxia. (B) MDA-MB 435 cells were incubated for approximately 20 hours until they were 50% confluent. Cells were then treated with RNAi to block HIF-1α expression. Cells were incubated for 24 hours in normoxic conditions, after which they were placed in anoxia for 16 hours or left in normoxia for 16 hours. Normoxic and anoxic cells were then analyzed by immunoblot for the indicated proteins. C indicates inverted control RNAi.