(A) Hectd3^–/– mice (n = 9) and littermate WT controls (n = 9) were infected subcutaneously with 3.0 × 10⁵ CFUs of F. novicida, and survival was monitored. (B) Body weight of WT and Hectd3^–/– mice after F. novicida infection as in A, presented relative to the starting body weight at day 0, which was set as 100%. (C) Hectd3^–/– mice and littermate WT controls were infected subcutaneously with 3.0 × 10⁵ CFUs of F. novicida, and bacterial burden in the spleen, liver, and lung on day 2 after infection was measured. (D) H&E staining of liver sections from WT and Hectd3^–/– mice on day 2 after infection with F. novicida. Arrowheads indicate infiltrated immune cells. (E) Ly-6G immunohistochemical staining of spleen and liver sections from WT and Hectd3^–/– mice on day 2 after infection with F. novicida. Arrowheads indicate neutrophil recruitment. Scale bars: 50 μm. (F and G) Expression of genes encoding TNF-α, IL-6, and IFN-β (F) and production of TNF-α, IL-6, and IFN-β (G) were analyzed in liver tissues from WT and Hectd3^–/– mice on day 2 after infection with F. novicida (F.n.). (H) Liver tissue samples from WT (W1, W2, and W3) and Hectd3^–/– mice (H1, H2, and H3) on day 2 after infection with F. novicida and uninfected WT (W) and Hectd3^–/– (H) mice were homogenized, and lysates were analyzed for activation and expression of caspase-3, caspase-1, caspase-11, ZBP1, and HECTD3, and phosphorylation of IκBα, STAT1, and STAT3. GAPDH was used as loading control. Each symbol indicates an individual mouse (C, F, and G). Data represent 3 independent experiments and are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.