BbtCaspase-8 interacted with DED of bbtFADD1 and induced HeLa cell apoptosis. (A) Comparison of the domains of bbtCaspase-8 and its mutants with those of dDREDD and hsCaspase-8. (B) Subcellular localization of bbtCaspase-8-GFP and its mutants. (C) Overexpression of bbtFADD1 did not co-localize with full-length of bbtCaspase-8, but co-localized with prodomain mutant C8-1. (D) Annexin V PE staining was analyzed (excitation at 488 nm and emission at 578 nm) from cells transfected with indicated plasmids. The third bar represented the sample in the presence of 50 μM z-IETD-fmk. The negative control was transfected with vector pCMV and the positive control was treated with camptothecin at 10 μM for 24 h. Apoptosis rates were expressed as annexin V positive cells/10,000 gated cells. (E) Caspase-8 activity in HeLa cells was measured after 20 h transfection with indicated plasmids. Data from experiments with hsCaspase-8 was presented as a positive control. All data shown were means ± standard deviations of three samples for each treatment and values were considered to be significant when P < 0.05. Results were confirmed by at least three separate experiments.