FIGURE 5.

A, effect of Peg3/Pw1 knockdown on β-catenin protein expression in D566 glioma cells using three different Peg3/Pw1 shRNAs. B, left panel, effect of three Peg3/Pw1 shRNAs on PEG3 mRNA expression in D566 glioma cells determined by real-time PCR. Data are means ± S.E. of four replicates (p < 0.01, t test). Middle panel, effect of Peg3/Pw1 knockdown on bromodeoxyuridine incorporation into DNA in D566 glioma cells overexpressing three separate Peg3/Pw1 shRNAs. Data are means ± S.E. of seven replicates (p < 0.03, t test). Right panel, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide growth assay showing the effect of Peg3/Pw1 knockdown on growth of D566 glioma cells overexpressing three separate Peg3/Pw1 shRNAs. Data are means ± S.E. of seven replicates (p < 0.02, t test). C, primary human CD133⁺ glioma-derived stem-like cells transduced with a control (Cont) or a β-catenin lentivirus and maintained as tumor spheres for 14 days. Scale bar = 80 μm. D, fluorescence micrographs showing β-catenin inhibition of DNA damage-induced apoptosis in glioma-derived stem-like cells revealed by annexin V staining of apoptotic cells (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). Cells were transduced with either a control lentivirus or a β-catenin lentivirus prior to exposure to camptothecin (CPT; 50 μm). The data are quantitated in right panel. E, β-catenin immunoreactivity in non-tumor brain (NTB) and Grade (GR) I–IV astrocytomas. Lower right panel, quantitation of β-catenin immunoreactivity in 29 astrocytomas of different grades (p < 0.04, t test). F, proposed role of Peg3/Pw1 in a p53/Siah1-dependent β-catenin (β-cat) degradation pathway.