Fig. 4

NLRP3 deletion blocked ROS-induced inflammasome activation during N. caninum infection in vitro. WT and Nlrp3^−/− PMs were pre-treated with or without the ROS inhibitor NAC (5 mM, 1 h), then infected with N. caninum at an MOI of 1 for 2 h, infected PMs were incubated with or without PG at post infection for 3 h. a ROS production was assessed using a multifunctional fluorometric reader. WT and Nlrp3^−/− PMs were pre-treated with or without the ROS inhibitor NAC (5 mM, 1 h), then infected with N. caninum at an MOI of 2 for 8 h, and incubated with or without PG. ATP (5 mM; 30 min) added to the N. caninum-infected cells was used as a positive control. At 36 h post-infection, IL-1β in supernatants was detected by ELISA (b), IL-1β (p17) and NLRP3 expression were detected by western blotting (c). WT and Nlrp3^−/− PMs were pre-treated with or without the ROS inhibitor NAC (5 mM, 1 h) or DPI (10 μM, 2 h), and infected with N. caninum at an MOI of 1 for 2 h, then cultured in fresh R-1% medium for 3 h, ROS production was assessed using a multifunctional fluorometric reader (d); or infected with N. caninum at the indicated MOIs for 8 h, and incubated with or without PG, at 36 h post-infection, IL-1β in supernatants was detected by ELISA (e). f Percent LDH release is measured by the LDH assay kit. The data are shown as the mean ± SE from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the Nc group in WT PMs or Nlrp3^−/− PMs; ^###P < 0.001 vs PG30-Nc (MOI = 2) in Nlrp3^−/− PMs