Figure 1

Effects of PKCδ knockdown by shRNA on proliferation and viability of human pancreatic (PCSC) and prostate (PrCSC) cancer stem cell cultures. (A) PCSC and PrCSC cells were grown to 50% confluence in 96-well plates and then infected with PKCδ-shRNA-expressing lentivirus vector or a lentiviral vector containing a scrambled shRNA (sc-shRNA). The corresponding equivalent volumes of diluent used for infection served as vehicle controls (Vehicle). 24 and 72 hr after transfection, cell mass was evaluated by MTS assay. Error bars represent SEM. p values for comparison between control (scrambled shRNA) and PKCδ-shRNA effects on cell number reached significance at 24 hr of exposure (p < 0.001) for all cell lines, and remained significant at the 72 hr time point. (B) PCSC and PrCSC cells were grown to 50% confluence in 96-well plates and then infected with PKCδ-shRNA or scrambled shRNA (sc-shRNA) expressing lentiviruses. The corresponding equivalent volumes of diluent were used as vehicle controls (Vehicle). After 24 and 72 hr of infection, cell cytotoxicity was evaluated by LDH-release assay. Total maximal LDH release was assigned the arbitrary value of 100% (Control). Error bars represent SEM. p values for comparison between effects on LDH release for cells infected with scrambled shRNA-expressing vectors compared to PKCδ-shRNA vectors reached significance at 24 hr of exposure (p < 0.01) for all cell lines, and remained significant at the 72 hr time point. (C) Immunoblot analysis of PKCδ protein levels in the same cell lines 72 hr after infection with PKCδ-targeting shRNA expressing lentiviral vectors (+) or scrambled shRNA (-). PKCδ-targeted shRNA vectors efficiently reduced PKCδ protein expression. Immunoblotting with a β-actin antibody after stripping the blots served as a loading control.