ASC^C171A leads to NLRP3 inflammasome-dependent hyperinflammation. (A) Immunoblot analysis for ASC oligomerization, pro–IL-1β, cleaved IL-1β, pro–caspase-1, and active caspase-1 p20 in LPS-primed Asc^wt and Asc^C171A BMDMs followed with or without ATP stimulation (representative of three independent experiments). (B) Production of IL-1β from LPS-primed Asc^wt and Asc^C171A BMDMs with or without of ATP or Oridonin treatment (n = 6 samples, representing three independent experiments; ND, not detected; **, P < 0.01; ***, P < 0.001 by two-tailed Mann–Whitney U test). (C) NLRP3 level and supernatant IL-1β from LPS-primed Asc^wt and Asc^C171A BMDMs with or without knockdown of Nlrp3, in the presence or absence of ATP stimulation (n = 3 samples, representing three independent experiments; ND, not detected; *, P < 0.05 by two-tailed Mann–Whitney U test). (D) IL-1β, IL-6, and TNF-α in sera from Asc^wt and Asc^C171A mice after LPS injection, with or without Oridonin treatment (n = 5 mice; ND, not detected; **, P < 0.01 by two-tailed Mann–Whitney U test). (E) Survival rate of Asc^wt and Asc^C171A mice after LPS injection (n = 10 mice; P = 0.0021 by log-rank test). (F–H) Picture of joints (F), the joint swelling gain (G), and IL-1β from joints and sera (H) of Asc^wt and Asc^C171A mice after injecting PBS or MSU for 24 h, with or without MCC950 treatment (n = 6 mice from two independent experiments; **, P < 0.01 by two-tailed Mann–Whitney U test).