Fig. 3.

Influence of PCa-relevant transcription regulators on NAMPT promoter and eNAMPT secretion. PC3 and Du145 cell cultures were treated with FG-4592 100 uM, or EGF 100 ng/ml, or testosterone 100 nM for 4 or 24 h. The NAMPT promoter activities (using dual-luciferase assay) were significantly increased in response to FG-4592 and EGF (p<0.05) as early as 4 h and were sustained for up to 24 h (A, B), results confirmed by western blots of NAMPT protein levels in cells at 24 h (C, D).

NAMPT promoter activities and expression were unchanged in response to testosterone challenge (A-D), Measurement of secreted eNAMPT into culture media of PC3 and Du145 cells (ELISA) were significantly increased in response to FG-4592 and EGF (p<0.05) but not to testosterone for 24 h (E, F). Detected by ChIP assay, HIF1α and HIF2α significantly bind to NAMPT promoter region under hypoxia (Hyp) compared to normoxia (Norm), and the bindings were significantly decreased by transfection of specific HIF1α SiRNA (siH1) or HIF2α  SiRNA (siH2) in both PC3 and DU145 cells (*, ** p<0.05). Non-specific IgG and siRNA (siC) were used as internal controls (G-H).