Fig. 2. Treatment with the broad-spectrum caspase inhibitor z-VAD-fmk protects cells from apoptotic cell shrinkage and activation of DEVDases but a decrease in mitochondrial TMRM uptake can still be detected. (A) MCF-7/Casp-3 cells stably expressing cyt-C-GFP and treated with 3 µM STS plus 200 µM z-VAD-fmk show a rapid cyt-C-GFP release and a decrease in TMRM uptake. Bar, 20 µm. (B) Individual traces of two cells treated with STS plus z-VAD-fmk. (C) Mean traces of 16 cells in four independent experiments calculated from single cell kinetics by setting the time of onset of cyt-C-GFP release to zero. (D,E) Transmission light and GFP fluorescence images of cells treated with 3 µM STS for the indicated time periods in the absence (D) or presence (E) of 200 µM z-VAD-fmk Bar, 20 µm. Note the absence of apoptotic shrinkage in z-VAD-fmk-treated cells despite cyt-C-GFP release. (F) z-VAD-fmk-insensitive decrease in TMRM uptake in HeLa D98 cells stably expressing cyt-c-GFP. Cells were treated with 3 µM STS in the presence of 200 µM z-VAD-fmk. Similar results were obtained in 14 cells in two separate experiments. (G,H) Simultaneous monitoring of DEVDase activity and TMRM uptake in single MCF-7/Casp-3 cells transiently transfected with the FRET probe CFP-DEVD-YFP and treated with 3 µM STS or 3 µM STS plus 200 µM z-VAD-fmk. The increase in the CFP/YFP ratio indicates the time course of the cleavage of the FRET probe. Similar kinetics of FRET probe cleavage and TMRM uptake could be detected in two independent transient transfection experiments per treatment. Traces in H have different time scales to demonstrate the absence of FRET probe cleavage up to 4 hours after completion of the TMRM fluorescence changes.