Cathepsin B is translocated from lysosomes into the cytosol during ATG-induced apoptosis. (A-B) PBLs were treated by NRS alone or ATG (250 μg/mL) in the presence or absence of indicated protease inhibitors. (A) The percentage of apoptotic cells was measured 24 hours later by surface binding of annexin V. Results are mean ± SEM from a triplicate experiment and are representative of 3 independent experiments. (B) DNA fragmentation was analyzed at 24 hours by using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. (C) Visualization of cathepsin B release from lysosomes into cytosol in PBLs treated by horse ATG (50 or 250 μg/mL). Staining with lysotracker red (acidic organelle-selective probe) was used to characterize lysosomal membrane alterations. (D) Cells were treated with rabbit or horse ATG (250 μg/mL), and cytosolic extracts were prepared at indicated times. Equal amounts of proteins were analyzed for cathepsin B activity using the fluorogenic protease substrate zRR-AMC. Results are mean ± SEM from a triplicate experiment.