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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. 7 — a, Cellular uptake of Sytox Green in THP-1 cells with indicated genotypes treated or not treated with 1 μg ml⁻¹ LPS or Pam3CSK4 for 2 h. Mean ± s.d., N = 3. ***P < 0.001. b, Immunoblotting of supernatants and cell lysates from THP-1 cells with indicated genotypes treated or not treated with 1 μg ml⁻¹ LPS in the absence or presence of 10 μM MCC950 or 20 mM KCl for 2 h. c, Cellular uptake of Sytox Green and ELISA analysis of cytokine secretion in experiments shown in b. Mean ± s.d., N = 3. ***P < 0.001; ns, not significant. d, Immunoblotting of culture supernatants and lysates from THP-1 cells. THP-1 cells with indicated genotypes expressing sgRNAs against ARFRP1 or SYS1 were treated with 1 μg ml⁻¹ LPS for 2 h. e, Confocal images of THP-1 cells with indicated genotypes treated with 1 μg ml⁻¹ LPS for 2 h stained with an antibody against ASC. Arrowheads indicate ASC specks. Scale bar, 20 μm. f, Quantification of ASC speck-containing cells in experiments shown in g. Mean ± s.d., N = 3, n = 100 cells for each group. ***P < 0.001. g, Immunoblotting of supernatants and lysates from THP-1 cells with indicated genotypes expressing GFP, Flag-SYS1, WT ARFRP1, T31N or Q79L mutant ARFRP1 treated with 1 μg ml⁻¹ LPS for 2 h. h, Cellular uptake of Sytox Green and ELISA analysis of cytokine secretion in experiments shown in g. Mean ± s.d., N = 3. ***P < 0.001; ns, not significant. i, Immunobloting of supernatants and lysates from THP-1 cells with indicated genotypes treated or not treated with FLn-Needle plus anthrax protective antigen or transfected with poly(dA:dT) for 4 h. j, Validation of generation of Arfrp1 KO and Sys1 KO iBMDMs. k, ELISA of IL-1β in supernatants from iBMDMs with indicated genotypes. After LPS for 4 h, cells were treated with 7.5 μM nigericin or 50 μM CL097 for 30 min. Mean ± s.d., N = 3. ***P < 0.001. Data were analyzed with an unpaired two-sided t-test (a, c, f, h, k). Data shown in b, d, e, g, i and j are representative of at least three independent experiments.

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