Fig. (3) Mitochondrial effects of casp8p41. (A) Characterization of casp8p41 induced swelling. Mouse liver isolated mitochondria were suspended in a hypoosmotic buffer, and their absorbance at 540 nm was recorded for 4000 sec at 37°C. Various doses of GST-casp8p41 were added in the presence of 10 µM Ca2+ (red line, 10 µM calcium, small dotted black line, 0.3 µM GST-casp8p41+10 µM calcium, large dotted black line, 1 µM GST-casp8p41 + 10 µM calcium, and black line, 3 µM GST-casp8p41 + 10 µM calcium). 100 µM Ca2+ (red dotted line) serves as a control of maximal swelling. The effect of GST (dark grey line) and GST-casp8p41 (grey line) was evaluated alone at the highest concentration (3 µM). Experiments were repeated three times. (B) Various inhibitors (CsA and Nfv) were added 2 min before the inducers, Ca²⁺ and GST-p41, at the indicated doses and their effect evaluated at 17 min. The effect of 3 µM GST-casp8p41 in the presence of 10 µM Ca²⁺ was normalized to 100%. Experiments were repeated three times and error bars represent SD. (C) Regulation of GST-casp8p41 induced Δγm loss. The Δγm loss was measured by the 123 Rhodamine dequenching method. Various inhibitors (CsA and Nfv) were added 2 min before the inducers, Ca²⁺ and GST-casp8p41, at the indicated doses. The effect of 3 µM GST-casp8p41 in the presence of 10 µM Ca²⁺ was normalized to 100%. Experiments were repeated three times and error bars represent SD. (D) GST-casp8p41 induced the cytochrome c, but not AIF release. Mouse liver isolated mitochondria were incubated in the presence of various agents and the release of cytochrome c, and AIF release was detected in the supernatant of mitochondria by Western-blot. Triton X-100 and alamethicin were used as positive controls of mitochondrial membrane permeabilization and Co. (untreated mitochondria) served as a negative control.