ROS promotes NLRP3 inflammasome activation via inducing GSTO1-mediated ASC deglutathionylation. (A and B) Coimmunoprecipitation of ASC and GSTO1 (A) and images of ASC-GSTO1 interaction (ASC-GSTO1 PLA; B) in unstimulated and LPS-ATP–stimulated BMDMs treated with or without Mito-TEMPO at indicated concentrations. Asc^−/− BMDMs were used as negative controls in B. Scale bars, 10 µm. (C) Numbers of ASC-GSTO1 PLA puncta in cells in B (n = 16–94 cells from three independent experiments; ****, P < 0.0001 by unpaired Student’s t test). (D and E) Immunoblot analysis for glutathionylated ASC (D) and images of glutathionylated ASC (ASC-GSH PLA; E) in unstimulated and LPS-ATP–stimulated BMDMs with or without Mito-TEMPO treatment at indicated concentrations. Asc^C171A BMDMs were used as negative controls in E. Scale bars, 10 µm. (F) Numbers of ASC-GSH PLA puncta in cells in E (n = 37–47 cells from three independent experiments; ****, P < 0.0001 by unpaired Student’s t test). (G and H) Numbers of ASC-GSTO1 PLA puncta (G) and ASC-GSH PLA puncta (H) in unstimulated and LPS-ATP–stimulated BMDMs with or without MitoQ or EN460 treatment at indicated concentrations (n = 51–125 cells from three independent experiments; ****, P < 0.0001 by unpaired Student’s t test). (I–L) Immunoblot analysis for ASC oligomerization, pro–IL-1β, cleaved IL-1β, pro–caspase-1, and active caspase-1 p20 in unstimulated and LPS-ATP–stimulated Asc^wt and Asc^C171A BMDMs, treated with or without Mito-TEMPO (I), MitoQ (J), combination of Mito-TEMPO and Oridonin (K), or EN460 (L). Data are representative of at least three independent experiments (A, B, D, E, and I–L).