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. 2021 Jul 28;11:15319. doi: 10.1038/s41598-021-94850-w

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© The Author(s) 2021, corrected publication 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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High-content imaging assay development. (a) Schematic of the assay protocol. (b) Untreated iBMDM cells show weak mCherry fluorescence, which strongly increases and localizes into a speck after inflammasome activation with LPS and nigericin (white arrows). Cell nuclei are visible in blue and ASC-mCherry in red. (c) ASC-mCherry iBMDM cells were treated with the indicated concentrations of MCC950 and LPS (1 µg/ml) for 2 h followed by nigericin treatment (10 µM, 2 h) after which PFA was added. The percentage of ASC specks/total nuclei normalised to LPS and nigericin is presented; (d) Immunoblot of pro-IL-1β and cleaved IL-1β (IL-1β p17), pro-caspase-1 (p45) and caspase-1 (p20) in the cell lysates and supernatants after cell stimulation with LPS + nigericin in the presence or absence of MCC950 (50 or 10,000 nM). β-actin was used as a loading control for the cell lysate samples. The IL-1β and caspase-1 bands are cropped from their respective blot images and the full-length blots are presented in Figure S5. Data in (c) are presented as mean ± SEM, n = 3 independent experiments and was analyzed using GraphPad Prism version 7 software (https://www.graphpad.com/scientific-software/prism/).