Figure 7

Absence of Apaf-1 prevented cell death and caspase-3 activation. (a) Confirmation of Apaf-1 knock-out cell and its wild-type counterpart by analyzing expression status of Apaf-1 by western blot. Apaf-1 protein was absent in knockout cells as shown in blot. (b) The cell death was assessed by nuclear condensation using Hoechst staining in MEF WT and MEF Apaf-1KO cells after PAC-1(50 μM) treatment. The condensed nuclei were counted and percentage has been plotted graphically relative to its control. Representative images and graph are shown. There was no sign of cell death in MEF Apaf-1KO cells while MEF WT cells were completely dead (n=4, mean ±S.D.; P<0.0001). (c) Caspase-3 cleavage was checked in both the cells after PAC-1 treatment by western blot. Caspase-3 cleavage was absent in Apaf-1 KO cell while wild-type cells showed the 17kDa band of cleaved caspase-3 clearly. (d) The cyt. c release was checked by immunofluorescence in MEF WT and Apaf-1 KO cells. Cyt. c release and nuclear condensation were massive in WT cells, but in Apaf-1 KO cells nuclear condensation was absent even though cyt. c released was noticed slightly after treatment for 24 h (scale bar: 20 μm)