FIG 1.

PRV infection induces IL-1β maturation and secretion in vitro. Mouse peritoneal macrophages isolated from C57BL/6 mice were infected with PRV at an MOI of 1, 5, or 10 for 12 h or infected with PRV at MOI of 1 for 3, 6, or 12 h as indicated. Mouse peritoneal macrophages were pretreated with LPS (1 μg/mL) for 8 h followed by treatment with 5 mM ATP for another 2 h, which was used as a positive control. The mRNA levels of IL-1β (A, D), IL-18 (B, E), and TNF-α (C, F) were detected by qPCR. The levels of secreted IL-1β in cell supernatants, PRV DNA copy number, and the protein levels of pro-IL-1β, IL-1β (p17), pro-caspase-1, and caspase-1 (p10) in cell lysates were detected by ELISA (G, H), qPCR (I, J), and Western blotting (K, L), respectively. (M, N) Mouse peritoneal macrophages were either mock infected or infected with PRV at an MOI of 1 for 12 h. The cell lysates were coimmunoprecipitated with anti-ASC monoclonal antibody (mAb) or mouse IgG. (M) The immunoprecipitants and the whole-cell lysates were detected with anti-NLRP3, AIM2, ASC, caspase-1, and GAPDH antibodies, respectively. Alternatively, the oligomerization of ASC was examined under confocal microscopy. The scale bars represent 10 μm in panel N. (O) The statistical analysis of ASC dots. The results shown are representative of three independent experiments (one-way ANOVA) in panels A to J. *, P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001. All error bars show standard deviations (SDs).