CSN5, but not CSN2, knockdown results in apoptotic cell death. K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and re-cultured. (A) Binding of Annexin V and uptake of propidium iodide were analysed by flow cytometry. The lower left quadrant encompasses the viable population of cells, the lower right quadrant contains early apoptotic cells, the upper right quadrant contains late apoptotic cells and the upper left quadrant contains the necrotic cell population. Dot plots shown are representative of n = 3 transfections. The mean of three data sets was taken and the values shown in the corresponding quadrant ± s.e.m. * indicates significance with a p value of less than 0.05. (B) Cells were harvested day 6 (shCSN2, top panels) or day 4 (shCSN5, bottom panels) post transfection and the level of caspase-9 cleavage determined by western blot. (C) Propidium iodide uptake was determined day 6 post transfection. The histogram shown is representative of n = 3 shVC (light grey in fill), shCSN2 (dark grey line) and shCSN5 (black line). The significant shift in propidium iodide staining in shCSN2 cells is shown ± s.e.m. * indicates significance with a p value of less than 0.05.
Pearce et al. BMC Cell Biology 2009 10:31 doi:10.1186/1471-2121-10-31