Figure 6
Ca2+-induced mitochondrial permeability transition pore opening and cytochrome c release are regulated by BI-1.
(A) Immunoblotting of total cells and mitochondria fractions from HT1080/Neo and HT1080/BI-1 with anti-Hsp60, anti-caspase-3, or anti-calreticulin antibodies. Mitochondria from HT1080/Neo and HT1080/BI-1 were treated with buffer and 100 μM CaCl2 in the presence or absence of 5 μM CsA. Absorbance at 540 nm was measured at the indicated time points after incubation at 30°C. Data shown are mean ± S.E. (n = 4). *p < 0.05 versus Ca2+-exposed mitochondria from HT1080/Neo. (B) Mitochondria from HT1080/Neo and HT1080/BI-1 were loaded with JC-1 dye and treated with 100 μM CaCl2 in the presence or absence of 5 μM CsA. Mitochondrial membrane potential (Δψm) was assessed using a spectrofluorometer as described in the Materials and Methods. (C) Mitochondria from HT1080/Neo and HT1080/BI-1 were treated with 100 μM CaCl2 in the presence or absence of 5 μM CsA. Cytochrome c released into supernatants was detected by immunoblotting with antibody against cytochrome c. Quantification is shown in the bottom panel. (D) Immunoblotting of total cells and mitochondria fractions from BI-1+/+ and BI-1-/- MEF with anti-Hsp60, anti-caspase-3, or anti-calreticulin antibodies. Mitochondria from BI-1+/+ and BI-1-/- MEF cells were treated with buffer, 100 μM CaCl2, or 100 μM CaCl2 plus 5 μM CsA. Absorbance at 540 nm was measured at the designated time points after incubation at 30°C. *, p < 0.05 versus Ca2+-exposed mitochondria from BI-1+/+ MEF cells, #, p < 0.01 versus Ca2+-exposed mitochondria from BI-1-/- MEF cells. (E) Mitochondria were loaded with JC-1 dye. Δψm was assessed using a spectrofluorimeter as described in the Materials and Methods. (F) Mitochondria were treated with 100 μM CaCl2 in the presence or absence of CsA. Cytochrome c released into the supernatant was detected by immunoblotting with antibody against cytochrome c. Quantification is shown in the bottom panel. *, p < 0.05 versus non-treated mitochondria from BI-1+/+ MEF cells, #, p < 0.01 versus non-treated mitochondria from BI-1-/- MEF cells. CsA: Cyclosporine A, Neo, neomycin-resistant pcDNA3-transfected HT1080 cells (HT1080/Neo); BI-1, HA-BI-1-pcDNA3-transfected HT1080 cells (HT1080/BI-1); BI-1+/+, BI-1 wild type mouse embryo fibroblasts; BI-1-/-, BI-1 knock-out mouse embryo fibroblasts. Sup, supernatants.
