Fig. 5. BRD7 destabilizes HIF1α through the ubiquitin-mediated degradation.

a The mRNA expression of BRD7, LDHA, HIF1α, and  β-tubulin were analysised by reverse transcription-PCR (RT-PCR) under hypoxia in BRD7-overexpressing cells. b Western blot analysis of BRD7/Flag, HIF1α, and β-tubulin  expression in MCF-7 and MDA-MB-231 cells transfected with pIRES2-EGFP-3Flag/BRD7 under hypoxia (1%O₂) for 24 h. c Western blot analysis of HIF1α and  β-tubulin expression in MCF-7 cells transfected with pIRES2-EGFP-3Flag/BRD7 and treated with CoCl2 (150 μM) and MG132 (10 μM) for the indicated periods of time (0, 4, 8, 12, 16 h) (left). Quantitative results also were shown (right). d MCF-7 cells transfected with pIRES2-EGFP-3Flag/BRD7 or pIRES2-EGFP for 24 h and treated with CoCl₂ (150 μM) for 24 h. Then the cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated periods of time (0, 1, 3, 6 h) (left). Lysates were harvested from the cells and analyzed by western blotting (left panel). Quantitation of HIF1α protein levels were shown in the right pane. e The MCF-7 cells transfected with pIRES2-EGFP-3Flag/BRD7 and pCDNA3.1-HIF1α were under hypoxia (1% O₂) for 24 h. Cellular co-localization of BRD7 and HIF1α in MCF-7 cells was analyzed by immunofluorescence staining with anti-BRD7 and anti-HIF1α under hypoxia (1% O₂) for 24 h. f MCF-7 cells transfected with the indicated pIRES2-EGFP-3Flag/BRD7 and pCDNA3.1-HIF1α were subject to immunoprecipitation (IP) respectively with anti-Flag antibodies (upper) and anti-HIF1α (down) under hypoxia (1% O₂) for 24 h. g The MCF-7 cells transfected with pIRES2-EGFP-3Flag/BRD7 and pCDNA3.1-HIF1α were under hypoxia (1%O₂) for 24 h before collection to immunoprecipitation (IP) with anti-HIF1α antibodies