(A) PC3 cells were grown in RPMI medium in the absence or presence of recombinant VEGF165 (50 ng/ml) for 24 hours. Cells were photographed and extracts were immunoblotted to assess expression of EMT markers. (B) Photomicrographs of PC3 cells that express either an empty vector (shCon), GFP shRNA (shGFP) or 2 different NRP1 shRNAs (shNRP1A and shNRP1B) were treated with or without TGF-β for 3 days. Extracts from these cells were immunoblotted for NRP1, as well as EMT markers. (C) Extracts from PC3 cells stimulated with either TGF-β, normoxia (N) or hypoxia (H), or ERβ1 knockdown cells (shERβ1) were immunoblotted with Abs specific for pAkt (Ser473), pGSK-3β (Ser9), Akt, GSK-3β and β-actin. (D) Extracts of LNCaP cells maintained in either normoxia (N) or hypoxia (H) for 24 hrs were immunoblotted with the same Abs. (E) PC3 cells were treated in the absence or presence of 3β-Adiol or PHTPP and subsequently analyzed for phospho-GSK3β, total GSK3β and Snail1 expression. Scale bars = 50 μm.