FTY720-mediated cell death is dependent on lysosomal membrane permeabilization and cathepsin activity. (A) To determine the relationship between lysosomal volume and cell death, Jeko-1 and Mino cells were treated with FTY720 or chloroquine at the indicated concentrations for 8 hours. Cells were then labeled with LysoTracker Green and costained with annexin V-PE. Changes in lysosomal volume (FL1) and cell death (FL2) were assessed by 2-channel flow cytometry. Representative histograms summarizing the percentage of LysoTracker⁺/annexin V⁺ cells are also shown. (B) Jeko-1 and Mino cells were treated with FTY720, chloroquine, rapamycin, or combinations at the indicated concentrations for 8 hours. Cells where then stained with AO (1 μg/mL) for 15 minutes. The relative changes in FL1 fluorescence were assessed by flow cytometry. Flow cytometric data from single experiments are shown in the left panels; representative histograms summarizing AO fluorescence intensity of MCL treated with FTY720, chloroquine, rapamycin, or combinations are shown in the right panels (MFI of treated cells is normalized to the untreated controls). (C) Jeko-1 and Mino cells were treated with FTY720 at the indicated concentration in the presence or absence of cathepsin inhibitor III (5 and 10μM). Cell death was determined by annexin V/propidium ioidie staining and flow cytometry at 8 hours. Data are shown as the percentage of annexin V⁻/propidium iodide⁻ cells (live cells).