Figure 7From: SMC3 knockdown triggers genomic instability and p53-dependent apoptosis in human and zebrafish cellsCell DNA content analysis and chromosomal abnormalities following SMC3 knockdown. A) Cell cycle FACS analysis of zebrafish embryonic cells. Embryos at 1–2 cell stage were injected with 1 nl Danieau buffer (left-hand side) or 4 ng of smc3-MO (center) or 4 ng of smc3-MO together with 250 pg smc3 mRNA (right-hand side). Batches of 30 embryos at 24 hpf were incubated in 0.05% trypsin 15 min at 25°C and the cells dissociated by pipetting through a large bore glass pipette. After fixation in 70% ethanol at 4°C overnight, cells were stained with propidium iodine/RNase, and the DNA cell content analyzed by flow cytometry. Gated data were used for analysis. Note the presence of a population of aneuploid (DNA > 4) cells in embryos following Smc3 knockdown. B) Analysis of the centrosome organization in human cells. Human 293 cells were transfected with 50 ng/ml of SMC3-siRNA and after 24 h plated on a poly-lysine-coated coverslip. Fourty-eight h later the cells were fixed in 4% para-formaldehyde, permebilized and then incubated with 1:1000 anti-human γ-tubulin antibody. Following incubation with Alexafluor 567-conjugated anti-rabbit IgG (Molecular Probes) and DNA staining with DAPI, the cells were examined under a fluorescence/UV microscope and the red (Alexafluor 567) and blue (DAPI) signals combined.Back to article page