Figure 5

HA-PTOV1 overexpression promotes invasion and anchorage-independent growth of PC-3 cells in contrast to the effects of exogenous Notch1 expression. (A) PTOV1 induces in vitro cell invasion, in contrast to the inhibition caused by Notch1. PC-3 cells were transiently transfected with 5 μg of the indicated plasmids and/or 5μg of DNA carrier (CTL) and treated with DAPT or solvent for three days before trypsinization and plating on Matrigel-coated Transwell wells. After 24 h, invading cells were stained with Hoechst and scored. Short hairpin sh1397 was used to knockdown PTOV1 expression. Assays were performed in triplicate in at least two independent experiments. (B) PTOV1 promotes anchorage-independent growth of PC-3 cells. Cells stably knocked down for PTOV1 grew significantly fewer spheroids than control cells. In contrast, cells stably expressing HA-PTOV1 formed significantly more spheroids than cells transduced with control lentivirus. (C) Notch1 overexpression significantly decreases anchorage-independent growth of PC-3 cells. Independent clones c4 and c15, with stable expression of Notch1 as shown by Western blotting, formed significantly fewer spheroids than control cells transfected with control pcDNA3 vector. S2 and S3 indicate partial and active forms of Notch1 receptor. * p < 0.05, ** p < 0.005, *** p < 0.0005.