Fig. 1 ~. Newly synthesized mtDNA is required for NLRP3 inflammasome activation.

a, Tfam^ΔMye BMDMs transduced with shRNA against Nlrp3, Aim2 (shNlrp3, shAim2) or control shRNA (shCtrl) and primed with LPS were incubated with synthetic mtDNA, ox-mtDNA, nuclear DNA (nDNA) or oxidized nuclear DNA (ox-nDNA), and the release of IL-1β (left) and TNF (right) was measured 4 h later. b, Relative total mtDNA amounts were quantified by quantitative PCR (qPCR) with primers specific for the mitochondrial D-loop region or a region of mtDNA that is not inserted into nuclear DNA (non-NUMT) and primers specific for nDNA (Tert, B2m) in wild-type BMDMs before and after LPS priming. c, Fluorescent microscopy of EdU-labelled newly synthesized mtDNA in wild-type BMDMs before and after LPS priming. Scale bars, 5 μm. Images are representative of three independent experiments. d, Relative total mtDNA amounts in wild-type BMDMs transduced with Polg shRNA (shPolg#1 and shPolg#2) or control shRNA, before and after LPS priming. e, IL-1β (left) and TNF (right) release by shCtrl-or shPolg-transduced LPS-primed BMDMs treated with different inflammasome activators. f, Relative total mtDNA amounts in wild-type (WT), Myd88^−/− and Trif^−/− (Trif is also known as Ticam 1) BMDMs after LPS priming. Data in a, b, d–f are mean ± s.d. (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001; two-sided unpaired t-test. NS, not significant.