LPS/ATP-induced association of NLRP3, ASC, and procaspase-1 requires DAPK in macrophages. (A) Formation of inflammasome measured by the association of NLRP3 and ASC in LPS/ATP-treated macrophages. Bone marrow-derived macrophages (2 × 105) were seeded overnight, treated without or with LPS, followed with or without addition of ATP. Macrophages were stained with anti-NLRP3 and anti-ASC antibody pair, or anti-NLRP3 and anti-caspase-1 antibody pair overnight at 4°C. Macrophages were then incubated with oligonucleotide-conjugated antimouse antibodies and antirabbit antibodies. The primers were then annealed, circularized, and amplified via rolling-circle amplification. Images were obtained on a Zeiss Axioplan 2 imaging microscope with objective lens of plan-Apochromat 63×/1.4 oil DIC at room temperature. Samples (fixed macrophages) were mounted on Duolink mounting medium. Texas Red–conjugated probe was detected using filter set of 598 nm (Ex) and 613 nm (Em), whereas DAPI-bound DNA was detected using filter set of 360 nm (Ex) and 460 nm (Em). The pinholes are Ch3-1:106 (red), Ch2-2:82 (blue), and ChD-3.0. Images were acquired by LSM 510 META (Zeiss) digital camera using Zeiss LSM Image Browser Version 4.2.0.121 software. For quantitation, individual amplicons were counted in 5 different fields and divided by the number of cells to obtain spots/nucleus. (B) Association of DAPK and NLRP3 in LPS/ATP-stimulated macrophages. Bone marrow-derived macrophages were treated as in panel A, incubated with anti-DAPK and anti-NLRP3, or anti-DAPK and anti–caspase-1, and the proximity of DAPK to NLRP3 detected as in panel A. ***P < .001 for paired t test.
