Fig. 3. F_OF₁-ATP-synthase reversal maintains _M^-cyt-C. (A) Addition of 10 µM FCCP after the release of cyt-C-GFP dissipates TMRM uptake. Cells were treated with 3 µM STS. FCCP was added approximately 2 hours after two cells had released cyt-C-GFP. (B) Total depolarization after the release of the cyt-C-GFP fusion protein in cells treated with 3 µM STS and 5 µM of the F_OF₁-ATP-synthase inhibitor oligomycin. The cells received 5 µM oligomycin 1 hour after addition of 3 µM STS. Note that mitochondria were not able to retain their TMRM fluorescence after the release of cyt-C-GFP and loss of fluorescence was followed by necrosis. Loss of the diffuse cyt-C-GFP fluorescence owing to plasma membrane rupture is indicated with white arrowheads. (C) Individual traces of two typical cells treated with 3 µM STS and 5 µM oligomycin, demonstrating that mitochondria were not able to retain TMRM following the release of cyt-C-GFP. (D) Mean traces of n=15 cells from three independent experiments were calculated by setting the time of cyt-C-GFP release to zero. (E) Addition of 5 µM oligomycin to cells 40 and 60 minutes after the release of cyt-C-GFP led to a rapid, permanent and total depletion of mitochondrial TMRM fluorescence. Similar results have been obtained in two separate experiments.