Figure 5

Role of p53 and BAX in SMC3-deficiency mediated apoptosis in human HCT116 cells. A) Western immunoblot assay of SMC3. p53-null, BAX-null and the matching wild-type parental HCT116 cells were transfected with 50 ng/ml SMC3-siRNA (5'-AACAGCGGUGGCUUUGC-3') and harvested 72 h later in lysis buffer. Proteins were separated on 7.5% SDS-PAGE. After transfer onto a nitrocellulose filter, SMC3 was detected using a goat anti-SMC3 antibody followed by ECL reaction. For normalization of the protein loading, the filters were stripped of the SMC3 immunocomplexes and reacted with anti-human β-actin antibody. Semiquantitative analysis of the protein level was performed by densitometric scanning of the autoradiographs. B) Flow cytometry of the cell DNA content. At 72 h post-transfection, cells were collected by trypsinization and fixed overnight in 70% ethanol at 4°C. After centrifugation, cells were washed once in PBS and then incubated with propidium iodide/RNase. Cell DNA fluorescence intensity was analyzed on a Coulter Epic flow cytometer. Results were gated and deconvoluted with ModFitLT to determine the fraction of apoptotic cells with < 2 N DNA fluorescence intensity. In the graphs the fraction of apoptotic cells is highlighted in black.