Fig. 2: The long IL of novel CgCaspase-3/6/7 inhibits its cleavage activation.

A Progress curves reporting on the purified CgCASP3 processing of the caspase-specific substrates (Ac-YVAD-pNA (caspase-1), Ac-VDQQD-pNA (caspase-2), Ac-DEVD-pNA (caspase-3/7), Ac-LEVD-pNA (caspase-4), Ac-VEID-pNA (caspase-6), Ac-IETD-pNA (caspase-8) and Ac-LEHD-pNA (caspase-9)) as a function of time in caspase activity buffer with 50 μM PAC-1 (n = 5). B Progress curves reporting on the purified CgCASP3 processing of the caspase-3/-7 substrate (Ac-DEVD-pNA) as a function of time in the presence of universal caspases inhibitor (Z-VAD-FMK) or caspase-3/-7 inhibitor Ac-DEVD-CHO (n = 5). C Coomassie staining of purified CgCASP3 and its long IL deletion mutant (CgCASP3-∆M) treated with 50 μM PAC-1 for 120 min. The cell-based transfection DEVD-ase activity assay (D.; n = 5), the CCK-8 (Cell Counting Kit-8) assay for the cell viability (E.; n = 5), the lactate dehydrogenase (LDH) release assay (F.; n = 5) and the cell apoptosis rate (G.; n = 3; the left panel is the cell apoptosis chart in each group, and the right panel is the cell apoptosis rate in each group) of HEK293T cells were transfected with mCherry-CgCasp3 or mCherry-CgCasp3-∆M, and incubated with TNF-α + SM-164 for 8 h. The control group is the HEK293T cells transfected with the pCMV-N-mCherry empty plasmid. H The western blotting of HEK293T cells transfected with mCherry-CgCasp3 or mCherry-CgCasp3-∆M, and incubated with TNF-α + SM-164 for 8 h. I Progress curves reporting on the purified CgCASP3 and CgCASP3-∆M processing of the caspase-3/-7 substrate (Ac-DEVD-pNA) as a function of time in caspase activity buffer with 50 μM PAC-1 (n = 5). The error bars represent the S.D. Significant differences among groups were marked with ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001. “ns” indicates non-significant differences.