Fig 2: PFOS-induced mtDNA accumulation triggers AIM2 inflammasome activation.a mtDNA amount from cytosolic extracts derived from THP-1-derived macrophages stimulated with PFOS was detected by qRT-PCR. b, c Relative enrichment of DNA in AIM2-pulldown material from Mock or PFOS (150 μM, 6 h) condition. qRT-PCR for three sets of primers that amplify fragments from different regions of the human genomic gDNA (b) or mtDNA (c). d mtDNA amount of total DNA extractions from THP-1-derived macrophages or bone marrow-derived macrophages (BMDMs) treated for 7 days without or with ethidium bromide (EtBr). e–h ELISA analysis of IL-1β secretion from supernatants of THP-1-derived macrophages (e, red plots) or BMDMs (f, blue plots) depleted of mtDNA and stimulated with PFOS (150 μM, 6 h), poly (dA:dT) (2 μg/ml, 6 h) or pre-treated with LPS (200 ng/ml, 3 h) followed by ATP (5 mM, 6 h). The cleavage of caspase-1 (p10) and the maturation of IL-1β (p17) in the cell supernatants or pro-Casp1 and pro-IL-1β in the cell lysates of THP-1-derived macrophages (g) or Bone marrow-derived macrophages (BMDMs) (h) depleted of mtDNA were determined by immunoblot. In a–f, all error bars, mean values ± SEM, P-values were determined by unpaired two-tailed Student’s t test of n = 3 independent biological experiments. For g and h, similar results are obtained for three independent biological experiments. Source data are provided as a Source data file.

Fig 3: ASC activates apoptosis in the absence of caspase-1 activity.(A) HEK 293T cells or HEK 293T cells ectopically expressing pro-caspase-1 C285A were transiently transfected with plasmids encoding RFP (mock, 2.0 μg), ASC (0.01 μg), CARD8UPA-CARD (0.02 μg), or CARD8UPA-CARD plus ASC. After 24 h, the cells were analyzed by immunoblotting. (B) HEK 293T cells were transiently transfected with plasmids encoding RFP (mock, 2.0 μg), ASC (0.01 μg), NLRP1UPA-CARD (0.02 μg), or NLRP1UPA-CARD plus ASC, or the indicated plasmids plus CASP1 C285A (0.01 μg), for 24 h and then the cell lysates were evaluated by immunoblotting. All data, including immunoblots, are representative of three or more independent experiments.

Fig 4: Phosphatidic acid (PA) binds to CytK and is necessary for CytK-induced pyroptosis.A A membrane lipid strip pre-coated with indicated lipids (left) were incubated with CytK and then probed with anti-His antibody (right). B, C dTHP-1 cells were pretreated with or without (−) an increasing dose of R59022 for 6 h, and then treated with or without (−) CytK (500 nM) or nigericin (Nig, 20 μM) for 1 h. LDH release in the cellular supernatants was determined (B). The supernatant together with the corresponding cell lysate were blotted with antibodies against Casp1, GSDMD, or β-actin (loading control) (C).

Fig 5: mtROS contributes to MRSA clearance in casp11 −/− macrophages MitoSOX assay in WT and casp11 −/− BMDMs (MOI 5:1) at 4 h post‐infection. Data represent mean ± SEM (n = 15 biological replicates). Statistical analysis was performed using multiple t‐tests. **P ≤ 0.01, NT = no treatment.Intracellular CFU of MRSA (MOI 5:1) in WT and casp11 −/− BMDMs treated with Ant A at 24 h post‐infection. Data represent mean ± SEM (n = 6 biological replicates). Statistical analysis was performed using two‐way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, NT = no treatment.Immunoblot analysis of cleaved CASP1 and IL‐1β in supernatants from WT and casp11 −/− BMDMs treated with Ant A at 24 h post‐infection (MOI 20:1). Densitometry analysis represents mean ± SEM (n = 4 biological replicates). Statistical analysis was performed using two‐way ANOVA. **P ≤ 0.01, NT = no treatment.