Lysosomal changes associated with GA101-induced cell death. (A) To determine changes in lysosomal volume, Raji cells were treated with mAbs (10 μg/mL) for 1 or 4 hours, labeled with Lysotracker green (75nM) and analyzed by flow cytometry. Histograms represent cells treated with control mAb (black), GA101 (blue), rituximab (purple), and unlabeled cells to set the background (solid red). GA101 induced an enlargement of the lysosomal compartment at 1 hour and a subsequent collapse in a subpopulation of the cells at 4 hours, whereas rituximab induced no changes in lysosomal volume. (B) To directly correlate cell death with lysosomal volume, cells were treated with mAbs. After 24 hours, cells were costained with Lysotracker green and annexin V Cy5.5 to label the dead cell population and analyzed by flow cytometry. Staurosporine (STSP) was used as a positive control for apoptosis. Cell death evoked by GA101 was associated with a collapse of the lysosomal compartment (upper left quadrant, in red). (C) Cells were preincubated with the V-ATPase inhibitor concanamycin A (CMA, 100nM) before treatment with mAbs and death was analyzed 24 hours after treatment. CMA significantly inhibited cell death induced by GA101 (*P < .001). (D) Cells were treated as above and lysosomal volume was quantified using Lysotracker green staining 1 hour after treatment. Histograms represent cells treated with control mAb (black), control mAb + CMA (purple), GA101 (blue), GA101 + CMA (green), and background (solid red). CMA prevents the increase in lysosomal volume induced by GA101.