FIG. 4.

CSA protects BL-3 cells against LKT-mediated cytotoxicity, mitochondrial membrane damage, and collapse of ψ_m. (A) BL-3 cells (10⁶ cells) were preincubated with CSA (2 or 5 μM) for 30 min before incubation with LKT (0.5 U) for 60 min. Cytotoxicity was measured using the Cell Titre 96 AQ one-assay system. (B) Mitochondria were immunopurified from untreated BL-3 cells, preincubated with CSA (5 μM) or with propranolol (Prop) (200 μM), and then incubated with LKT (0.2 U) for 30 min at 37°C. Immunoblot analysis of the resulting mitochondrial lysates demonstrated that CSA, but not propranolol, protected against LKT-mediated mitochondrial cytochrome c (Cyt c) release. Con, control. (C and D) BL-3 cells were preincubated with CSA or medium (control) and then incubated with LKT (0.5 U) for 1 h. Cells were then incubated with JC-1, a ψ_m-sensitive dye, at 37°C for 15 min and washed three times in PBS. Aggregated intramitochondrial JC-1 is red, and the monomeric cytoplasmic form is green. BL-3 cells were then analyzed by flow cytometry in the FL2 channel (absorption and emission maxima at 580 and 595 nm, respectively). The results show that CSA protected against collapse of ψ_m. The bars in panel D indicate the means of three separate experiments, and the error bars indicate the standard errors of the means. *, P < 0.05.