FIGURE 1.
Rotenone promotes NLRP3 inflammasome activation only with ATP costimulation. A, mouse BMDMs were untreated (Unt) or treated with LPS (0.25 μg/ml) for 3 h, followed by treatment with CCCP (40 μm) or rotenone (Rot) (10 μm) for 2 h, or were treated with CCCP or rotenone for 2 h, followed by treatment with ATP (2.5 mm, 30 min), as indicated. B, mouse BMDMs were treated with CCCP or rotenone (1–20 μm) for 2 h, followed by treatment with ATP. C, mouse BMDMs were treated with LPS in the presence of CCCP or rotenone (1–10 μm) for 2 h, followed by ATP treatment. D and E, primary WT or Nlrp3−/− BMDMs (D), or immortalized WT, Nlrp3−/−, or Asc−/− BMDMs (E) were untreated or treated with LPS or rotenone (10 μm) for 3 h, followed by treatment with ATP. F, mouse BMDMs were treated with LPS or rotenone (5 μm) for 3 h, followed by ATP, nigericin (Nig), or alum treatment, as indicated under “Experimental Procedures.” A–F, culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies.