Fig. 1. Release of cyt-C-GFP triggers a decrease in mitochondrial TMRM uptake. (A) MCF-7/Casp-3 cells stably expressing cyt-C-GFP were equilibrated with 30 nM TMRM and treated with 3 µM STS. Cyt-C-GFP release was followed by a reduction in mitochondrial TMRM uptake. Bars, 20 µm. (B) Individual traces of two typical cells treated with 3 µM STS. The release of cyt-C-GFP was detected as a reduction in the standard deviation of the GFP pixel intensities of single cells. Changes in TMRM uptake were calculated from single cells by determining their average pixel intensity in the TMRM-sensitive channel. Squares and diamonds indicate corresponding TMRM and cyt-C-GFP changes of the two cells, respectively. The onset of the decrease in mitochondrial TMRM uptake occurred simultaneously to mitochondrial cyt-C-GFP release, suggesting an irreversible _M depolarization during the time period monitored. (C) Quantification of TMRM fluorescence decrease in the STS-treated cells. The time of cyt-C-GFP release was set to zero to calculate average values from 16 cells in four independent experiments. (D) Individual traces of two typical cells treated with 100 ng/ml TNF and 1 µg/ml CHX. Similar traces were recorded from 12 cells in two independent experiments represented.

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