Figure 4
Mitochondrial membrane potential loss precedes cyt. c release and caspase activation. (a) MCFC3 cells expressing cyt. c-EGFP were stained with TMRM, and 2 h after drug treatment imaged for both TMRM and EGFP as described. The representative images are shown (scale bar: 10 μm). The TMRM intensity is reduced before cyt. c release. Also, the pixel intensity profile of TMRM (red line) and cyt. c- EGFP (green line) of the indicated cell is shown. This graph and image clearly indicate that (Δψm) loss preceded cyt. c release from mitochondria. (b) SiHa FRET DEVD probe-expressing cells were seeded in 96-well glass-bottom plate and stained with TMRM before treatment with drugs. Drugs were added in medium containing low concentration of TMRM and images were captured with montage (2x2) at different time points using BD pathway 435 imager to read out large number of cell population. Scatter plot of FRET ratio and TMRM intensity after treatment with PAC-1 are shown. The shifting of population to left side suggests the loss in Δψm. Arrow marks represent the cells with FRET loss as well as Δψm loss. There was no cell detected with FRET loss without Δψm loss. (c) MCF7 cells expressing FRET DEVD probe were also used for determining FRET loss and TMRM intensity by BD pathway imager as described above. The arrow marks represent the FRET lost cell populations, which also reflected Δψm loss
