Figure 4

Knocking down CSNK1E induces G2/M cell cycle arrest and caspase-mediated apoptosis. (a) Two days after non-targeting shRNA or shCSNK1E treatment, HT1080 cells were fixed in methanol and stained with propidium iodide (Materials and methods). Flow cytometry of cells was performed on a FACSCalibur; calculation of cell cycle stages was performed using the cell cycle analysis program Modifit LT. Red area shows cell population in G1 or G2 cell cycle phase, while gray area shows dying cells. Label 'A' denotes apoptotic cell population. Insets show photographs of HT1080 cells treated with non-targeting shRNA or shCSNK1E. (b) Knocking down CSNK1E down-regulates CyclinB1 and CyclinA2. Cellular RNAs were prepared from HT1080 cells infected with either non-targeting shRNA (N.T.) or two different CSNK1E-targeting shRNAs (1834, 1837), and real-time PCR was performed with each gene-specific primer set. The expression levels of CyclinB1, CyclinA2 and CyclinD1 were first normalized to the level of an endogenous control (RPLPO), and then the relative expression level of each gene among the three cell lines was expressed as a ratio of transcripts in a cell line to those in non-targeted shRNA treated cells. Error bars indicate one standard deviation of triplicate data. (c) Knocking down CSNK1E induces caspase activation. Whole cell lysates from HT1080 cells infected with either non-targeting shRNA or shCSNK1E and cells treated with staurosporine were prepared. The cleavage of PARP1 or caspase-3 (Casp-3) in each sample was examined by western blotting using antibodies against PARP1 and cleaved caspase-3.