Fig. 6

The ability of the dam complementation strain and overexpression strain cultured without IPTG to activate the inflammasome was improved. C57BL/6 BMDMs were pre-treated with LPS (1 μg/mL) for 5 h (untreated and uninfected BMDMs was used as a negative control, Blank LPS-), and then infected with C50336, C50336Δdam, C50336Δdam::dam, C50336Δdam-pMMB207, C50336::dam, or C50336-pMMB207 at an MOI of 20 for 4 h uninfected BMDMs was used as another negative control (Blank LPS+). Bacteria bearing pMMB207 plasmids were cultured without IPTG. a. The ratio of cell death was evaluated by the release of LDH in the supernatants. b. The expression of caspase-1, NLRP3, NLRC4, ASC, and pro-IL-1β were analyzed by immunoblotting. β-actin was blotted as a loading control. Molecular mass markers in kDa are indicated on the right. Original images of immunoblotting were shown in Fig. S6. The production of IL-1β c and IL-6 d in the supernatants were examined via ELISA. ***p < 0.001 for one-way ANOVA followed by Bonferroni’s multiple comparison test indicate significant findings in comparison with cells infected with WT strain C50336. Data are presented as mean ± SEM of triplicate samples per experimental condition from three independent experiments