Ectopic overexpression of PTOV1 downregulates endogenous HEY1 and HES1 . (A) Cells were transduced with lentivirus for HA-PTOV1 (black bars) or control lentivirus (white bars) and analyzed for the expression of HES1 and HEY1 by real-time RT-PCR. Inlet: Western blotting for the detection of ectopic HA-PTOV1 (slower migrating band in transduced cells) and endogenous HES1. (B) The transcriptional repressor activity of PTOV1 is downstream of Notch receptor processing. PC-3 cells, transfected with the HES- luciferase and TK-Renilla reporter plasmids, were co-transfected with ΔE (partially processed) or ICN (fully processed) forms of Notch1 without, or with HA-PTOV1, and transactivation of the HES1-promoter determined by luciferase assays. Statistical significance: * p < 0.05, ** p < 0.005.