Fig. 3

PG inhibited N. caninum replication by increasing NLRP3 inflammasome activation in vitro. PMs were treated with PG at various concentrations (15, 30, 60 and 80 μM) for 24 or 48 h. a The cell viability of PMs was determined by CCK-8 assays. b PMs were treated with PG (15 or 30 or 60 μM) for 3 h, PMs stimulated with zymosan (1 mg/ml; 1 h) was used as positive controls, ROS production in PMs was assessed using a multifunctional fluorometric reader. PMs were infected with N. caninum at the indicated MOIs for 2 h, and incubated with or without PG for 3 h. c ROS production was detected by a multifunctional fluorometric reader. PMs were infected with N. caninum at the indicated MOIs for 8 h, then incubated with or without PG. At 36 h post-infection, d cell death was monitored by measuring LDH activity in the supernatants, e IL-1β in supernatants was measured using ELISA, f NLRP3, IL-1β (p17) and caspase-1 (p20) were monitored by western blotting. PMs were infected with N. caninum at the indicated MOIs for 4 h, then incubated with or without PG. At 24 h post-infection, percentage of N. caninum infected cells (g) and quantification of parasites (h) in parasitophorous vacuoles were monitored by fluorescence microscopy. i Number of N. caninum in infected PMs was determined by qPCR. The data are shown as the mean ± SE from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the cell; ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001 vs Nc (MOI = 1); ^#P < 0.05, ^##P < 0.01 vs Nc (MOI = 3); ^aP < 0.05, ^aaP < 0.01 vs Nc (MOI = 2); ^bbP < 0.01 vs PG30 μM-Nc (MOI = 1)