Skip to main content
. Author manuscript; available in PMC: 2011 Apr 13.
Published in final edited form as: Cancer Cell. 2010 Apr 13;17(4):319–332. doi: 10.1016/j.ccr.2010.02.030

Figure 4. ERβ1 Destabilizes HIF-1α Protein and Represses HIF-1-mediated transcription of VEGF-A.

Figure 4

(A) PC3 cells maintained in either normoxia (N) or hypoxia (H) for 24 hrs, treated with PBS (Con) or TGF-β, transfected with control or ERβ1 shRNA or siRNA, or treated with PHTPP were analyzed for the expression of HIF-1α by immunoblotting. * denotes a non-specific band. HIF-1α mRNA was detected by RT-PCR in TGF-β–stimulated cells and shRNA transfected cells. (B) PC3 cells (scrambled control cells) or ERβ1 knockdown cells were treated in the absence or presence of MG132 (1 μM), 3β-Adiol (1 μM) or E2 (10 nM) for 6 hours and immunoblotted for HIF-1α. (C) PC3 cells (scrambled control cells) or ERβ1 knockdown cells were treated in the absence or presence of MG132 (1 μM) for 6 hours and photographed. Scale bars = 50 μm. Extracts of the control cells treated with MG132 were immunoblotted for E-cadherin, vimentin and β-actin. (D) PC3 cells expressing a scrambled shRNA (Scr) or an ERβ1 shRNA (shERβ1) were analyzed for VEGF-A mRNA expression by qPCR (left graph). PC3 cells were treated with PBS (Con) or TGF-β in the absence or presence of 3β-Adiol (1 μM) or 3β-Adiol (1 μM) plus PHTPP (5 μM). After 3 days, cells were analyzed for VEGF-A mRNA expression by qPCR (right graph). (E) VEGF-A secretion in culture medium from PC3 cells treated with PBS (Con) or TGF-β or transfected with control or ERβ1 shRNAs was quantified by ELISA. (F) Scrambled control cells (Scr) or ERβ1 knockdown cells (shERβ1) were transfected with a VEGF promoter reporter construct and luciferase activity normalized to Renilla was measured (left graph). PC3 cells were transfected with a wild type VEGF promoter reporter construct in the absence (Wt) or presence of TGF-β (WT+TGFβ). Concurrently, cells were transfected with the reporter construct containing either a mutated ERE (EREm) or both a mutated ERE and HRE (EREm/HREm) and normalized luciferase activity was measured (right graph). (G) Scrambled control cells (Scr) or ERβ1 knockdown cells (shERβ) were transfected either with a wild-type HRE reporter construct: Scr (Wt) or shERβ (Wt) or with a mutated version of the HRE reporter construct: Scr (mut) or shERβ (mut) under hypoxic conditions for 16-18 hours and normalized luciferase activity was measured. All data are the mean of 3 separate experiments with SEM and P value (*) < 0.05 indicated.