(a) J774A.1 macrophages exposed to EtBr (0, 10, 100 and 500 ng/ml) were stimulated with LPS and ATP. Cytokine secretion was analyzed by ELISA. Lysates and supernatants were immunoblotted for caspase-1 and IL-1β. (b) J774A.1 or ρ0 macrophages (EtBr; 100 ng/ml) were incubated with LPS, followed by ATP (15 min and 30 min). Lysates were immunoblotted for caspase-1 and ASC. (c) J774A.1 l or ρ0 macrophages were incubated with LPS, and ATP. Cytokine secretion was analyzed. (d) LPS-primed macrophages were incubated with rotenone (5 μM) or DMSO 1 h before ATP. Cytokine secretion was analyzed. Lysates were immunoblotted for caspase-1 and IL-1β. (e) J774A.1 or ρ0 macrophages were incubated with LPS and rotenone, followed by ATP. IL-1β secretion was analyzed by ELISA (f) Peritoneal macrophages pre-incubated with Mito-TEMPO for 1 h were stimulated with LPS and ATP. Cytokine secretion was analyzed. (g,h) Peritoneal macrophages isolated from Map1lc3b−/− (g) or Becn1+/− (h) mice were incubated with Mito-TEMPO (500 μM) for 1 h, followed by LPS and ATP. IL-1β secretion was analyzed by ELISA. Lysates were immunoblotted for caspase-1. *P < 0.05 (Student's t-test). Data represent three experiments (mean and s.d. in a,c,d,e,f,g,h).