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. 2018 Oct 12;9:4246. doi: 10.1038/s41467-018-06568-5

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© The Author(s) 2018

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. 4 — Rev-erbα inactivates Nlrp3 inflammasome at the priming step. a qPCR measurements of Nlrp3, IL-1β, ASC, and caspase-1 in Raw264.7 cells. The cells were pretreated with Rev-erbα agonist (10 μM), TCDD (10 nM, as a positive control), or vehicle for 1 h and then stimulated with LPS for 8 h. Data are mean ± SD (n = 3). *P < 0.05 versus DMSO-treated group (Mann–Whitney U test). b Measurements of Nlrp3 mRNAs in Raw264.7 cells by qPCR. The cells were pretreated with DMSO or a series of concentrations of Rev-erbα agonist (SR9009, hemin, or GSK4112) for 1 h and then stimulated with LPS for 8 h. c Measurements of Nlrp3 mRNAs in Raw264.7 cells by qPCR. The cells were pretreated with DMSO or SR9009 (10 μM) for 1 h and then stimulated with LPS for indicated hours. d Protein expressions of Nlrp3, pro-IL-1β, ASC, and pro-caspase-1 in Raw264.7 cells measured by western blotting. The cells were pretreated with DMSO or SR9009 (10 μM) for 1 h and then stimulated with LPS for 12 h. e mRNA expressions of Nlrp3, IL-1β, ASC, and caspase-1 in Raw264.7 cells measured by qPCR. The cells were transfected with Rev-erbα siRNA or Rev-erbα plasmid for 24 h and then stimulated with LPS for 8 h. f Protein expressions of Nlrp3, Pro-IL-1β, ASC, and pro-caspase-1 in Raw264.7 cells measured by western blotting. The cells were transfected with Rev-erbα siRNA or Rev-erbα plasmid for 24 h and then stimulated with LPS for 12 h. g Western blotting of Nlrp3, cleaved caspase-1 (p20), cleaved IL-1β (p17), pro-caspase-1, and pro-IL-1β in the supernatants (Sup) or cell lysates (Lys) of PMs. PMs were pretreated with SR9009 or vehicle for 1 h and then stimulated with LPS for 12 h and ATP for 30 min (added last). The concentrations of LPS and ATP for cell treatment were 100 ng/ml and 2 mM, respectively. Each western blot is representative of three independent experiments (statistical differences between blot density levels were analyzed by Mann–Whitney U test, Supplementary Figure 12). In panels c and e, data are mean ± SD (n = 3), *P < 0.05 (Mann–Whitney U test). Casp1: caspase-1