Fig. 3: The phosphorylation of Thr260 at CgCaspase-3/6/7 inhibits its activation.

A The phosphorylation levels of Thr260 site of CgCASP3 in gill tissues of C. gigas and C. angulata during heat stress, which was obtained from our previous study³⁰. B The DEVD-ase activity assay of C. gigas and C. angulata during heat stress (n = 3). C The sequence alignment surrounding T260 on CgCASP3. The cell-based transfection DEVD-ase activity assay (D.; n = 5), the CCK-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, mCherry-CgCasp3^T260A (mimicking dephosphorylation) or mCherry-CgCasp3^T260D (mimicking phosphorylation), 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. There were no significant differences among the four groups in panels (D) (0, 2, 4 h) and E (8 h). H The western blotting of HEK293T cells transfected with mCherry-CgCasp3, mCherry-CgCasp3^T260A or mCherry-CgCasp3^T260D, and incubated with TNF-α + SM-164 for 8 h. I Progress curves reporting on the purified CgCASP3, CgCASP3^T260A and CgCASP3^T260D 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.