Cathepsin B-specific inhibitor suppresses Y570C-induced cell death and Y570C-transfection induces lysosomal leakage. (A) Y570C-transfected THP-1 cells were analyzed by flow cytometry 3 hours after transfection. Cells undergoing death are represented as cells that are smaller in size on the FS axis, and positive for annexin V/7-AAD. The cathepsin B–specific inhibitor CA-074-Me (50 μM) was added to THP-1 cells just after Y570C transfection. The number in each quadrant shows the percentage of cells. (B) Small-scale lysosomal leakage (ssΔLL) following transfection of WT-CIAS1 and Y570C lacking the GFP epitope tag was assessed by incubating cells with 1 μg/mL of acridine orange and monitoring fluorescence using confocal laser microscopy. In Y570C-transfected cells, cytosolic red fluorescence was reduced, accompanied by a dimming of the green-fluorescent structures of the nuclei. The top scale bar represents 20 μm; the bottom represents 5 μm. (C) The percentage of the cells with ssΔLL 3 hours after transfection with either WT-CIAS1 or Y570C. Error bars indicate SD (n = 6). (D) Analysis of ssΔLL by flow cytometry in cells transfected with the indicated constructs, in the presence or absence of 50 μM cathepsin B–specific inhibitor CA-074-Me. The cathepsin B–specific inhibitor CA-074-Me (50 μM) suppressed ssΔLL in Y570C-transfected cells. ssΔLL represents the loss of red fluorescence, and each number shows the percentage calculated in the region under the bar. (E) The percentage of cells with ssΔLL assessed by flow cytometry. Transfection with Y570C resulted in a time-dependent increase in ssΔLL in THP-1 cells. Representative data from 3 independent analyses of similar results are shown.