β-catenin-induced mitochondrial fission triggers cyt c release, activating the caspase-1/IL-1β/caspase-3 axis in A. hydrophila-infected ZKM. (A) ZKM pre-treated with or without β-catenin inhibitor (JW67), Drp1 inhibitor (Mdivi-1), and MPTP inhibitor (CsA) for 1 h were infected with A. hydrophila, and changes in cytosolic cyt c were studied at 12 h p.i. (B) ZKM pre-treated with β-catenin inhibitor (JW67), caspase-1 inhibitor (Z-YVAD-FMK), Drp1 inhibitor (Mdivi-1), and MPTP inhibitor (CsA) for 1 h were infected with A. hydrophila, and relative changes in caspase-1 activity were studied at 12 h p.i. mtROS inducer (Ant A) was used as a positive control in this study. (C) ZKM pre-treated with β-catenin inhibitor (JW67), MPTP inhibitor (CsA), Drp1 inhibitor (Mdivi-1), and caspase-1 inhibitor (Z-YVAD-FMK) for 1 h or transfected with sc-siRNA, il1b-siRNA were infected with A. hydrophila, and relative changes in IL-1β concentration were plotted at 12 h p.i. (D) ZKM pre-treated with or without β-catenin inhibitors (JW67 and MSAB), NOX inhibitors (Apo and DPI), mtROS inhibitor (YCG063), Drp1 inhibitor (Mdivi-1), MPTP inhibitor (CsA), caspase-1 inhibitor (Z-YVAD-FMK), and caspase-3 inhibitor (Ac-DEVD-CHO) for 1 h or transfected with sc-siRNA, il1b-siRNA were infected with A. hydrophila, and relative changes in caspase-3 activity were studied at 24 h p.i. Additionally, ZKM were incubated with canonical Wnt/β-catenin pathway activators (LiCl and Laduviglusib) and Ant A, and relative changes in caspase-3 activity were studied at 24 h p.i. Vertical bars denote the mean ± SEM (n = 3). Asterisk (*) denotes a significant difference between the indicated groups (* p < 0.05). “+B” mentioned in the X-axis represents “+A. hydrophila”.
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