npsn deficiency affects host defence against E. coli infection. (a) The infection site of the zebrafish yolk sac (the red arrow). (b) Scheme showing the experimental procedure used for survival assays. The npsnsmu5 mutants and WT controls were infected with E. coli at 2 dpf via the yolk sac, and the number of surviving larvae was counted daily over the next 5 days. At least three independent experiments were performed using greater than 60 embryos per group. (c) Survival curves of WT embryos challenged with different doses of E. coli. Mortality increased in a dose-dependent manner. (d) Survival curves of npsnsmu5 and WT embryos injected with 100 CFUs of E. coli (WT (n = 57); npsnsmu5 (n = 55) in total). Statistical significance was determined by the log-rank test. *p < 0.05. (e) Bacterial burden of embryos injected with E. coli. Significantly more bacterial cells were observed in npsnsmu5 mutant embryos when compared with those observed in WT controls at 1 and 2 dpi. Data were combined from three individual experiments (n = 50 per group), and statistical significance was determined using the two-way ANOVA with Bonferroni's multiple comparisons adjustment. *p < 0.05. ***p < 0.001. n.s., not significant. #, undetected. (f) Alteration of the inflammatory response in npsnsmu5 embryos at 2 hpi. The relative quantity of inflammatory factors il-1b, il-8 and tnfα was examined by qRT-PCR, and expression levels were adjusted for trauma (PBS-solution injection). (Mean ± s.e.m., n = 30 in each group, triplicated). Statistical significance was determined using the one-way ANOVA with Bonferroni's multiple comparisons adjustment. *p < 0.05. **p < 0.01. ***p < 0.001. n.s., not significant.
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