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Fig. 1 Macrophages are required for Shigella training and contribute to zebrafish survival (A, D, and G) Schematic of the zebrafish immune training model in the HBV in the absence of macrophages using IRF8 zebrafish mutants (A) and MTZ treatment during the 1st infection (D) or 2nd infection (G). (B) Survival rates of IRF8+/+ and IRF8−/− zebrafish larvae (N = 3 with >8 larvae per experiment). Left graph represents collated data and right graph represents only IRF8−/− larvae, a subset of the data on the left. (C) Bacterial burden of naive and Shigella-trained (IRF8+/+ 1.1 × 103 ± 8.3 × 102 colony-forming units [CFUs] and IRF8−/− 1.3 × 103 ± 7.6 × 102 CFUs) larvae upon secondary infection (IRF8+/+ PBS 2.6 × 104 ± 7 × 103 CFUs, IRF8+/+ Shigella 2.7 × 104 ± 7.5 × 103 CFUs, IRF8−/− PBS 2.7 × 104 ± 7.5 × 103 CFUs, and IRF8−/− Shigella 2.3 × 104 ± 6.8 × 103 CFUs). (E) Survival rates of DMSO- and MTZ-treated zebrafish larvae (N = 3 with >18 larvae per experiment). Left graph represents collated data and right graph represents only MTZ-treated larvae, a subset of the data on the left. (F) Bacterial burden of naive and Shigella-trained (DMSO 8 × 102 ± 2.4 × 102 CFUs and MTZ 1.2 × 103 ± 4 × 102 CFUs) larvae upon secondary infection (DMSO PBS 2.6 × 104 ± 8 × 103 CFUs, DMSO Shigella 2.7 × 104 ± 1.1 × 104 CFUs, MTZ PBS 2.9 × 104 ± 1 × 104 CFUs, and MTZ Shigella 3.3 × 104 ± 1.2 × 104 CFUs). (H) Survival rates of DMSO- and MTZ-treated zebrafish larvae (N = 3 with >14 larvae per experiment). Left graph represents collated data and right graph represents only MTZ-treated larvae, a subset of the data on the left. (I) Bacterial burden of naive and Shigella-trained (1.3 × 103 ± 6.8 × 102 CFUs) larvae upon secondary infection (PBS DMSO 2.1 × 104 ± 1.5 × 104 CFUs, Shigella DMSO 2.5 × 104 ± 4.7 × 103 CFUs, PBS MTZ 2.2 × 104 ± 8.8 × 103 CFUs, and Shigella MTZ 2.7 × 104 ± 5.1 × 103 CFUs). ns, non-significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.01, and ∗∗∗∗p < 0.0001. Error bars represent standard deviation (SD). (B, E, and H): log rank (Mantel-Cox) test. (C, F, and I) Two-way ANOVA with Tukey’s multiple comparisons test.