Y570C-transfection induces mitochondrial damage. (A) Loss of mitochondrial inner transmembrane potential (Δψm) was assessed by flow cytometry using 1 μM of MitoTracker DeepRed 633. Δψm is represented as the loss of deep red fluorescence. The cathepsin B–specific inhibitor CA-074-Me (50 μM) suppressed Δψm in Y570C-transfected cells. Each number shows the percentage calculated in the region under the bar. (B) The percentage of cells with Δψm increased in a time-dependent manner following transfection with Y570C. (C) Full-length caspase-3 was detected by using a caspase-3 antibody, and cleaved caspase-3 was detected by using a cleaved caspase-3–specific antibody. Y570C-tranfection resulted in slight cleavage of caspase-3. However, this level of cleavage was far less than that induced by actinomycin D (ActD) treatment, but similar to the level induced by nigericin treatment. (D) After transfection with the WT-CIAS1 or Y570C mutant (3 hours), THP-1 cells were harvested and caspase-3 activity was measured according to the manufacturer's protocol. THP-1 cells treated with 1 μg/mL ActD and 20 μM nigericin (Ni) were used as controls. The fold increases in caspase-3 activity are the means of the normalized data (no treatment = 1) of triplicate cultures, and error bars indicate SD. (E) Neither cleaved caspase-8 nor Bid cleavage was observed upon Western blot analysis of cells following Y570C transfection. Bid cleavage can be assessed by the loss of full length of Bid. HeLa cells without treatment or treated with 20 ng/mL TNF-α and 10 μg/mL cycloheximide (TNF + CH) were used as positive controls for protein cleavage. (F) Y570C transfection did not induce caspase-1 cleavage at least 3 hours after transfection. Representative data from 3 independent analyses of similar results are shown.