β-catenin plays a pro-apoptotic and anti-bacterial role in A. hydrophila-infected ZKM. (A) 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 % Hoechst-positive ZKM were enumerated at 24 h p.i. Additionally, ZKM were incubated with canonical Wnt/β-catenin pathway activators (LiCl and Laduviglusib) and Ant A for 1 h and % Hoechst-positive ZKM were enumerated at 24 h p.i. (B) ZKM pre-treated with or without β-catenin inhibitors (JW67 and MSAB) for 1 h were infected with A. hydrophila and stained with AV-PI at 24 h p.i. Next, % apoptotic ZKM were calculated. Similarly, ZKM were incubated with canonical Wnt/β-catenin pathway activators (LiCl and Laduviglusib) for 1 h and % Hoechst-positive ZKM were enumerated at 24 h p.i. (C) ZKMs 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 the bacterial loads were enumerated at 24 h p.i. Additionally, ZKM were incubated with canonical Wnt/β-catenin pathway activators (LiCl and Laduviglusib) and Ant A for 1 h and % Hoechst-positive ZKM were enumerated at 24 h p.i. Vertical bars represent the mean ± SEM (n = 3). Asterisk (*) and hash (#) denote a significant difference between the indicated groups (* p < 0.05). “+B” mentioned in the X-axis represents “+A. hydrophila”.
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