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. 2022 Aug 4;1(8):732–747. doi: 10.1038/s44161-022-00108-7

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© The Author(s), under exclusive licence to Springer Nature Limited 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

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Extended Data Fig. 7 — a, In vitro, FACS analysis displaying the caspase 1 activity in platelets (Tlr4^-/- or WT) treated with 1 μg/ml rmS100A8/A9 for 4 hours (n = 5). The quantified results are shown on the right. b-f, In vivo, a total of 1.2 ×10⁷ purified platelets (volume: 200 μl, concentration: 6 ×10¹⁰ platelets/L) from Tlr4^-/- or WT mice were intravenously transfused to mT/mG: PF4-Cre mouse. (b) After platelet depletion, platelet counts in mice were assessed at 0, 2, 4, 6, 8, 24 and 26 hours using a hematology analyzer (n = 3). (c) Platelet counts in mice before and after transfused with WT or Tlr4^-/- platelets were detected at 0, 0.5, 2, 4 and 6 hours using a hematology analyzer (n = 3). (d) The percentages of transfused Tlr4^-/- platelets in total platelets of mice were detected at 0, 0.5, 2, 4 and 6 hours using FACS analysis (n = 3). (e) Caspase 1 activity was measured using FACS analysis (n = 6). (f) The association of ASC and NLRP3 inflammasome in murine platelets was measured by immunofluorescence analysis. Platelets were stained for CD41 (green), ASC (red) and NLRP3 (blue); scale bars: 5 μm and 1 μm; n = 6. Data was presented as mean ± SD. One-way ANOVA and Tukey’s multiple comparisons test for a. Two-way ANOVA test for c, d. Abbreviation is as follow: HBSS, hank’s balanced salt solution; Saline, mice transfused with normal saline; WT PLTs, LPS-injected mice transfused with WT platelets; Tlr4^-/- PLTs, LPS-injected mice transfused with Tlr4^-/- platelets. PBS, PBS-injected mice; LPS + saline, LPS-injected mice transfused with normal saline; LPS + WT PLTs, LPS-injected mice transfused with WT platelets; LPS + Tlr4^-/- PLTs, LPS-injected mice transfused with Tlr4^-/- platelets; LPS, lipopolysaccharide; NLRP3, NOD-like receptors containing domain pyrin 3 inflammasome; ASC, adaptor-apoptosis-associated speck-like protein.

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