In situ association of NLRP3 and ASC in macrophages is PML-dependent as measured by proximity ligation assay. (A-B) In situ association of NLRP3 and ASC in macrophages activated by LPS and ATP. BMDMs were plated overnight and sequentially stimulated with LPS and ATP as indicated. Macrophages were stained overnight at 4°C with anti-NLRP3 (Cryo-2, mouse MAb) and anti-ASC (AL177, rabbit pAb), followed by oligonucleotide-linked anti-mouse antibodies and anti-rabbit antibodies. After the addition of the template oligonucleotides, the primers in close proximity were allowed to anneal and circularize. The products generated by rolling-circle amplification were detected by hybridization with a Texas red–conjugated probe and were examined under a confocal laser scanning microscope (A). Amplicons were counted in at least 40 cells and divided by the number of cells to obtain spots/ν (nucleus) (B). (C-D) Close proximity between NLRP3 and caspase-1 in activated macrophages. The association of NLRP3 with caspase-1 in macrophages was analyzed (C) and quantitated (D) as in (A) and (B). (E-G) In situ association of PML with NLRP3 or caspase-1 in macrophages activated by LPS and ATP. BMDMs were treated with LPS and ATP, stained with anti-PML (H-238, rabbit pAb) and anti-NLRP3, or with anti-PML and anti-caspase-1, and the association of PML to caspase-1 or NLRP3 was measured (E) and quantified (F-G). The bar represents 10 μm. *P < .05; **P < .01 for paired t-test.