The role of lysosomal membrane permeabilization and lysosomal cathepsins in GA101-induced cell death. (A) Raji cells were incubated with acridine orange (AO) to label lysosomes, washed twice, treated with mAbs, and analyzed at different time points. Leakage of lysosomal contents into the cytoplasm was measured as an increase in green fluorescence detected by FACS. GA101 induced an increase in green fluorescence at 2 hours, followed by a subsequent loss of fluorescence at 4 and 8 hours. (B) Fluorescence microscopy of the lysosomal protease cathepsin B staining (red) of Raji cells 4 hours after treatment with mAbs. DNA was counterstained with DAPI (blue; scale bar, 40 μm). GA101 induces marked cathepsin B release into the cytosol and surrounding points of cellular adhesion. (C) Western blot of the active 25-kDa subunit of cathepsin B (CTSB) in cell supernatants 4 hours after treatment with mAbs. BSA was used as a loading control on the same supernatants diluted 1:5. GA101 evokes extracellular cathepsin B release (Ctl indicates control mAb; GA, GA101; and Rit, rituximab). (D) Cells were preincubated with cathepsin inhibitor III (100μM) before treatment with mAbs and cell death was analyzed 4 hours after treatment. Figure shows mean + SEM of triplicates, representative of 2 independent experiments. Cathepsin inhibitor III inhibits GA101-induced cell death (*P < .001).