Figure 1

Cell-based model to study kinetics of cyt. c release. (a) SW480 cyt. c-EGFP-expressing cells were treated with PAC-1 and after 3 h, time-lapse imaging was done by live-cell fluorescent microscopy at time intervals of 30 min. The representative images are shown (scale bar: 10 μm. SW480 cells were treated by PAC-1 for indicated time points and cyt. c release was checked by immunoblot in cytosolic fraction after digitonin permeabilization. (b) Caspase-3 lacking MCF7 cells were stably transfected with cyt. c-EGFP and treated with PAC-1 after staining with Hoechst. Live cell time-lapse imaging was done by fluorescent microscopy with a time interval of 15 min. Representative images of indicated time points are shown (scale bars: 10 μm). Diffuse pattern indicated the cyt. c release from mitochondria to cytosol, which is initiated around 8 h but nucleus was not condensed even up to 16 h. The cyt. c release was also checked by western blot in cytosolic fraction of PAC-1 treated MCF7 cells after digitonin permeabilization. (c) Caspase-3-expressing MCFC3 cells were stably transfected with cyt. c- EGFP and treated with PAC-1 after Hoechst staining. Time-lapse imaging was done at time intervals of 20 min. Representative images are shown (scale bars: 10 μm) and the results were also substantiated by western blot in MCF C3 cells after drug treatment as earlier. The fragmentation of cells after cyt. c release is rapid in caspase-3-expressing cells compared with MCF7. The arrows represent the condensed nucleus after cyt. c release. But, the initiation of cyt. c release was almost at same time point in both the cells (around 8–9 h)