Figure 3.

GR regulates HIF-2α expression and GR/HIF-2α are co-recruited to the Brk promoter. (A) MDA-MB-231 cells cultured in normoxia or hypoxia for 24hrs with vehicle, 1μM dex, 1μM RU486 or both agents and mRNA levels were assessed by qRT-PCR after normalization to TBP levels. Asterisks indicate statistical significance from all other treatment groups in either normoxia or hypoxia. (B) MDA-MB-231 cells were treated for 24hrs with vehicle, 1μM dex, hypoxia or both agents and subjected to Western blot analysis for HIF-1α, HIF-2α or ERK1/2 (loading control); Brk mRNA levels were analyzed by qRT-PCR after normalization to TBP levels. (C) Hs578T cells treated with vehicle or 1μM dex for 24hrs and mRNA expression was analyzed via qRT-PCR following normalization to TBP expression. (D) MDA-MB-231 cells were cultured at normoxia or hypoxia and treated with vehicle or 1μM dex for 1hr and ChIP assays were performed. Negative isotype-matched IgG controls were conducted on hypoxia and dex treated MDA-MB-231. (E) MDA-MB-231 cells were treated with vehicle or 1μM dex and hypoxia for 1hr. ChIP-Re-ChIP assays were performed with initial immunoprecipitation with GR antibody and subsequently immunoprecipitation with HIF-2α antibody. No secondary antibody was included in control samples to demonstrate specificity. Representative examples from triplicate experiments are shown. (*p<0.05, **p<0.01, ***p<0.001; unpaired Student t test).