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. 2022 Nov 28;24(1):30–41. doi: 10.1038/s41590-022-01355-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — a, Immunoblotting of supernatants and lysates from primary BMDMs isolated from Arfrp1^fl/fl or Arfrp1^fl/fl;LysM-Cre (Arfrp1^ΔMye) mice. Isolated cells were primed with 1 μg ml⁻¹ LPS for 4 h, followed by treatment with 2 mM ATP, 5 μM nigericin, 50 μM CL097 or 50 μM R837 for 40 min as indicated. Antibodies recognizing both p45 and p20 fragments of caspase-1 (CASP1), p31 and p17 fragments of IL-1β, NLRP3 and ARFRP1 were used. An antibody against tubulin was used as a loading control. Short exposures (s.e.) and long exposures (l.e.) of IL-1β and CASP1 fragments are shown. b, ELISA assay of IL-1β and TNF-α in supernatants of isolated BMDMs from mice treated as described for a. Mean ± s.d., N = 3. Data were analyzed with an unpaired two-sided t-test. c, Survival curves of Arfrp1^fl/fl or Arfrp1^fl/fl;LysM-Cre (Arfrp1^ΔMye) mice injected with LPS (15 mg kg⁻¹) in the presence of vehicle or 50 mg kg⁻¹ MCC950. The survival of mice after LPS injection was monitored every 6 h. n = 8 for vehicle-treated Arfrp1^fl/fl mice group; n = 8 for MCC950-treated Arfrp1^fl/fl mice group; n = 10 for vehicle-treated Arfrp1^ΔMye mice group and n = 9 for MCC950-treated Arfrp1^ΔMye mice group. Data were analyzed with a log-rank (Mantel–Cox) test. d, ELISA assay of IL-1β (left panel) and TNF-α (right panel) levels in serum collected from Arfrp1^fl/fl or Arfrp1^fl/fl;LysM-Cre (Arfrp1^ΔMye) mice at 3 h after peritoneal injections of LPS (15 mg kg⁻¹) in the presence of vehicle or 50 mg kg⁻¹ MCC950. Mean ± s.d., n = 8 for vehicle-treated Arfrp1^fl/fl mice group; n = 8 for MCC950-treated Arfrp1^fl/fl mice group; n = 10 for vehicle-treated Arfrp1^ΔMye mice group and n = 9 for MCC950-treated Arfrp1^ΔMye mice group. Data were analyzed with a two-sided Mann–Whitney U-test. e, Proposed model of NLRP3 inflammasome activation as described in Discussion. Data shown in a are representative of three independent experiments.

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