Fig. 3.

PHD2 sustains epithelial differentiation. (A) PHD2 ablation promotes dedifferentiation. PHD2-ablated cells (shPHD2#1 and shPHD2#2) and control cells (shGFP) were maintained in either 2D or 3D cultures. Note that the morphology of PHD2-ablated cells is similar to that of ERβ-ablated cells in Fig. 1. (Scale bars, 100 μm.) Immunoblot analysis of these cells indicates that loss of PHD2 induces a decrease in E-cadherin expression and an increase in mesenchymal markers. (B) Inhibition of PHD2 activity induces epithelial dedifferentiation. PNT1a cells were incubated for 2 d with DMSO (control) or 100 µM DMOG, a PHD2 inhibitor. DMOG-treated cells exhibit a mesenchymal phenotype with concomitant increase in vimentin and HIF-1α expression compared with controls. (C) PHD2 ablation induces epithelial dedifferentiation and promotes HIF-1α activity in LNCaP cells. PHD2-ablated LNCaP cells (shPHD2#1 and shPHD2#2) exhibit a mesenchymal phenotype compared with control cells. Loss of PHD2 expression also induces HIF-1α and vimentin expression. These cells were transfected with a reporter gene carrying either a wild-type HRE or a mutated HRE. A significant induction in luciferase activity is seen in PHD2-ablated cells compared with control cells with the wild type HRE-reporter gene but not with the mutant HRE reporter (*P < 0.05). (D) Inhibition of PHD2 activity induces dedifferentiation in LNCaP cells. LNCaP cells were incubated with either DMSO (−) or 100 µM DMOG (+) for 2 d. DMOG-treated cells exhibit a mesenchymal morphology and an increase in vimentin and HIF-1α expression compared with control cells.