Figure 5

The caspase activator PAC-1 blocks the negative regulation of zebrafish BIRC2 on the E. piscicida infection. (A) PAC-1 inhibited the zebrafish BIRC2-mediated bacterial proliferation at 24 hpi. (B) PAC-1 blocked the zebrafish BIRC2-mediated bacterial proliferation at 48 hpi. For (A, B), larvae were collected at 24 and 48 hpi, and used for colony count. Data represented the means ± the SEM (n = 3) and were tested for statistical significance using a two-tailed student’s t -test. **p < 0.01. The asterisk above the error bars indicates statistical significance using the group microinjected with empty plasmid and without treatment as the control group. The asterisk above the bracket indicates statistical significance between the two groups connected by the bracket. (C) The effect of PAC-1 on the survival rates of zebrafish larvae microinjected with p3×FLAG or BIRC2-FLAG in the case of E. piscicida infection. Exposures were performed in triplicate with 20 larvae per repetition (n = 60). Statistical differences using the log-rank test were observed between the FLAG/BIRC2-FLAG groups (p < 0.0001), FLAG/FLAG+PAC-1 groups (p < 0.05), and BIRC2-FLAG/BIRC2-FLAG+PAC-1 groups (p < 0.0001). (D–K) The effect of PAC-1 on the expression levels of apoptosis-related genes and NF-κB genes in zebrafish larvae microinjected with p3×FLAG or BIRC2-FLAG at 48 hpi. For (D–K), larvae were collected at 48 hpi, and used for qRT-PCR. Data represented the means ± the SEM (n = 3) and were tested for statistical significance. *p < 0.05; **p < 0.01. For (A–K), 4 dpf larvae microinjected with the p3×FLAG or BIRC2-FLAG were treated by adding PAC-1 for 12 h, and then infected with E. piscicida.