ox-DNA is released upon inflammasome activation. (A) Cell-free ox-DNA levels were analyzed by ELISA in THP1, SKM1, and U937 cells treated with lipopolysaccharide (LPS), adenosine triphosphate (ATP), and nigericin (collectively referred to as LAN), demonstrating increased ox-DNA in stimulated vs untreated (Untx) controls (mean ± SD of 3 independent experiments). (B) Confocal IF showing increased cytosolic ox-DNA in LAN-stimulated cells compared with untreated controls (DAPI; ox-DNA/RNA; magnification ×2520). Images are representative micrographs. (C) Western blot of CRISPR knockout (KO) cells of NLRP3 (top band), full-length caspase-1 (Casp1), and ASC. The lack of cell-free ox-DNA released was analyzed by ELISA after treatment with LAN (mean ± SD of 3 independent experiments). (D) Decreased BrdU (green) incorporation in rho0 cells by IF (DAPI, blue). (E) PCR amplification of nuclear DNA (nucDNA) or mitochondrial genes of DNA isolated from rho0 and parental cell lines. (F) ox-mtDNA levels assessed by ELISA in parental and rho0 cells after treatment with LAN or rhS100A9 (mean ± SD of 3 independent experiments). Scram, scrambled.