Figure 3.

PCDH-PC expression reduces ligand-bound AR activity. (A) Western blot analysis 48 hours following transient transfection of PCDH-PC cDNA or the control vector. (B) PSA promoter activity was assessed in transfected LNCaP cells by measuring luciferase activity 24 hours after DHT treatment in cellular extracts normalized to β-Gal activity. (C) Stable transfectants of vector- and PCDH-PC-transfected LNCaP (LNCaP-PCDH-PC) were examined for differences in cell morphology and cell growth (DT), and PSA promoter activity (D) as in B. (E) Western blot made against proteins from LNCaP-pcDNA3, LNCaP-AI, and LNCaP-PCDH-PC cells showing reduced PSA and increased levels of NSE in the LNCaP-PCDH-PC cells. (F) LNCaP-PCDH-PC cells were treated for 3 days with either the PI3K inhibitor LY294002 (10 µM) or vehicle (DMSO). A Western blot was performed and probed as above. (G) 22Rv1 cells transfected either with siRNAs raised against PCDH-PC mRNA or non-targeting siRNA were analyzed for mRNA expression of PCDH-PC, KLK3, and KLK2. Down-regulation of PCDH-PC is accompanied by elevation of KLK2 mRNA but had minor effects on KLK3. (H) 22Rv1 cells were treated with vehicle (EtOH) or DHT (10 nM) for 24 hours, and endogenous levels of KLK3 and KLK2 were examined. (I) 22Rv1 cells pretransfected with PCDH-PC plasmid were treated with vehicle (EtOH) or DHT (10 nM) for 24 hours, and PCDH-PC, KLK3, and KLK2 levels were compared by qPCR.