Fig. 4. Simultaneous depolarization of mitochondrial and plasma membrane potential during STS-induced apoptosis. (A) Changes in TMRM and Dibac₄(3) uptake in a typical MCF-7/Casp-3 cell after and exposure to valinomycin (10 nM, _M depolarization) and ouabain (100 µg/ml, _P depolarization). Similar traces were recorded in 10 cells in two experiments. (B) Individual traces of the average fluorescence intensity of two typical MCF-7/Casp-3 cells equilibrated with the positively charged TMRM and the negatively charged Dibac₄(3) after treatment with 3 µM STS. (C) Mean values of Dibac₄(3) and TMRM fluorescence kinetics were calculated by setting the time of onset of decrease in TMRM fluorescence to zero. Data represent 11 individual cells from three independent experiments. (D) Inhibition of caspases protects MCF-7/Casp-3 cells from STS-induced _P depolarization. Individual traces of two typical cells treated with 3 µM STS and 200 µM z-VAD-fmk. (E) Mean values of 12 cells treated with 3 µM STS and 200 µM z-VAD-fmk in two independent experiments. (F). Time course of Na⁺/K⁺-ATPase ß-subunit degradation during STS-induced apoptosis. Cells were treated with STS and whole cell extracts were analyzed by immunoblotting. (G) Treatment with z-VAD-fmk inhibits the degradation of the Na⁺/K⁺-ATPase ß-subunit and the activation of effector caspase-3. Cells were treated with 3 µM STS, 3 µM STS plus 200 µM z-VAD-fmk or vehicle. Whole cell extracts were prepared after 8 hours of treatment and analyzed by immunoblotting.