Figure 2

A. hydrophila-induced ZKM death is apoptotic in nature. (A) ZKM pre-treated with or without the caspase-3 inhibitor (Ac-DEVD-CHO) were infected with A. hydrophila (MOI 1:50), and apoptosis was assessed by enumerating Hoechst-positive ZKM at 24 h p.i., A(i), A(ii), and A(iii) represent the uninfected ZKM, A. hydrophila-infected ZKM, and A. hydrophila-infected ZKM pre-treated with Ac-DEVD-CHO, respectively, at 24 h p.i. (×40). Three different fields having at least 100 cells were observed for determining the percentage of Hoechst-positive ZKM. (B) B(i) represents uninfected ZKM, B(ii) A. hydrophila-infected ZKM, and B(iii) A. hydrophila-infected ZKM pre-treated with the caspase-3 inhibitor (Ac-DEVD-CHO), respectively, were stained with AV-PI at 24 h p.i., and photomicrographs were captured using the fluorescence microscope (×40). Three different fields having at least 100 cells were observed for determining the percentages of AV⁺PI⁺, AV⁻PI⁺, and AV⁺PI^- cells. (C) Uninfected and A. hydrophila-infected ZKM were lysed at 24 h p.i., with their gDNA then isolated and subjected to agarose gel electrophoresis alongside the 100 bp DNA marker. (D) ZKM were infected with A. hydrophila, and relative caspase-3 activity was measured at indicated time points p.i. Vertical bars denote 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”.