Fig 1: F. novicida mutant strain XWK4 enhances inflammatory cytokines expression and cell death. (A) BMDMs from WT mice were infected with F. novicida WT strain (U112, 20 MOIs) and mutant strain (XWK4, 20 MOIs) for 3 h, and intracellular bacteria were enumerated at indicated times. (B) Transmission EM analysis of U112- and XWK4-infected (100 MOIs) BMDMs for 2 h. Asterisk indicates the individual intracellular bacteria. Arrow indicates the vesicle that contains multiple bacteria. Scale bars: 2 µm for left and 0.5 µm for right. (C) Immunoblot analysis of TFEB and LC3 in uninfected and U112- and XWK4-infected (100 MOIs) BMDMs at indicated times. (D) Expression of Il1a, Il6, Cxcl9, Cxcl10, Isg15, and Mx1 was analyzed by qRT-PCR in U112- and XWK4-infected (100 MOIs) BMDMs for indicated times. (E,F) Cell death was analyzed by microscope (E) and LDH release assay (F) for U112- and XWK4-infected (200 MOIs) BMDMs (14 h). Arrow indicates the dying cell. Scale bars: 20 µm for (E). Each symbol indicates individual sample for (F). *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant (two-sided Student’s t-test without multiple comparisons correction). Data are representative of three independent experiments.