Effects of SOX2-RNAi on cell proliferation, cell cycle and induction of apopotosis in U343-MG and U373-MG glioma cells. a: Propidium iodide-staining and cell cycle analysis of U343-MG and of U373-MG cells 5 days after transduction with shRNA vectors revealed no induction of apoptosis (SubG1 fraction) but a decrease of cell fractions in S/G2/M-phases. As controls, shLuc transduced cells were included. As positive control for apoptosis U343-MG and U373-MG wild type cells were treated with doxorubicin to induce apoptosis (Wt+Dox). b: Growth kinetics of U343-MG and U373-MG cells transduced with shSOX2 #788, shSOX2 #2378, and shLuc (control). Note the gradual decrease in cell numbers in glioma cells with knockdown of SOX2. Bars in a: and b: indicate standard deviation. * p < 0.05 when SOX2-silenced cell were compared to shLuc controls. c: Western blot analysis of SOX2 and of cell cycle regulator proteins cyclinD1, cyclinE, retinoblastoma protein (pRB) and of pRB-pT356, indicating phosphorylation by the cyclinD1-cdk4 complex. Note the decreases in cyclinD1 expression and the concomitant diminished levels of pRB-pT356 in cells with knockdown of SOX2. d: Western blot analysis showing no cleavage of pro-caspase 3 and no increase of p21waf/cip inhibitor of cyclin-dependent kinases Cdk4/6 in U343-MG and U373-MG cells transduced with shSOX2 #2378 and in controls transduced with shLuc. (+) depicts U343-MG cells treated with doxorubicin serving as positive control for induction of apoptosis and induction of p21waf/cip. Equal loading of lanes in c: and d: was verified using tubulin antibody.