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. 2024 Jan 12;15(1):36. doi: 10.1038/s41419-023-06414-7

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© The Author(s) 2024

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 — A Immunoblots of NLRP3 expression in microglial whole-cell, cytoplasmic, and nuclear lysis after CMI, n = 12 mice per group. Quantitative ELISA analysis of TNF-α (B), IL-6 (C), IL-1β (D) and IL-10 (E) expression in the contralateral side of CMI showing that CMI did not influence the inflammatory cytokines expression in the chronic phase of CMI. (n = 12 mice per group). One-way ANOVA F _{4, 55} = 8.251, p < 0.0001. Sham vs. CMI1w: p = 0.0867; Sham vs. CMI 2w: p = 0.9064; Sham vs. CMI 3w: p < 0.0001; Sham vs. CMI 4w: p = 0.0331 (B); F _{4, 55} = 3.113, p = 0.0222. Sham vs. CMI1w: p = 0.9992; Sham vs. CMI 2w: p = 0.9211; Sham vs. CMI 3w: p = 0.0390; Sham vs. CMI 4w: p = 0.9554 (C); F_{4, 55} = 10.62, p < 0.0001. Sham vs. CMI1w: p = 0.2167; Sham vs. CMI 2w: p = 0.5763; Sham vs. CMI 3w: p = 0.9177; Sham vs. CMI 4w: p = 0.0027 (D); F_{4, 55} = 4.098, p = 0.0056. Sham vs. CMI1w: p = 0.4233; Sham vs. CMI 2w: p > 0.9999; Sham vs. CMI 3w: p = 0.0266; Sham vs. CMI 4w: p = 0.9984 (E) with Tukey’s correction. F Representative immunofluorescence images of NLRP3 (red) expression in microglia (iba1, green) one week after CMI. Scale bars, 20 μm. G Immunoprecipitation and immunoblot analysis of MLL1 and NLRP3 in microglial lysates from mice subjected to sham and CMI. Lysates were immunoprecipitated by anti-MLL1 (IP) or immunoglobulin control (IgG) or directly subjected to immunoblot (Input), n = 3 mice per group. H Proximity ligation assay of microglial (iba1, green) NLRP3 and MLL1 (red dots) in sham and one-week post-CMI mice. Scale bars, 20 μm. I ChIP-seq analysis of NLRP3 and MLL1 binding to IL-1β in one-week post-CMI mice. J Representative images of sequence alignments showing interaction of NLRP3 (blue) with MLL1 (green). K Immunprecipitation and immunoblot analysis of MLL1 and Flag in microglial lysates from Cxc3r1^CreER mice transfected with 3xFlag-NLRP3 or truncated mutants (ΔPYD, ΔNACHT, ΔLRR) and subjected to CMI surgery. Lysates were immunoprecipitated by anti-MLL1 (IP) or immunoglobulin control (IgG) or directly subjected to immunoblot (Input), n = 3 mice per group. Quantitative analysis of H3K4me3 ChIP-qPCR on TNF-α (L), IL-6 (M), IL-1β (N), and IL-10 (O) promoters in contralateral microglia one week after CMI. Cxc3r1^CreER mice transfected with 3xFlag-NLRP3 or truncated mutants (ΔPYD, ΔNACHT, ΔLRR) and subjected to CMI surgery. One-way ANOVA, F _{4, 55} = 42.25, p < 0.0001;Empty vs Full Length, p < 0.0001; Full Length vs ΔPYD: p = 0.7675; Full length vs ΔNACHT, p < 0.0001; Full Length vs ΔLRR: p = 0.1030 (L); F _{4, 55} = 8.732, p < 0.0001; Empty vs. Full Length, p = 0.0077; Full Length vs ΔPYD: p = 0.9998; Full length vs ΔNACHT, p = 0.0001; Full Length vs. ΔLRR: p = 0.3921(M); F _{4, 55} = 122.5, p < 0.0001;Empty vs. Full Length, p < 0.0001; Full Length vs ΔPYD: p = 0.9868; Full length vs. ΔNACHT, p < 0.0001; Full Length vs. ΔLRR: p = 0.2638 (N); F _{4, 55} = 2.246, p = 0.0758; Empty vs Full Length, p > 0.9999; Full Length vs ΔPYD: p = 0.9988; Full length vs ΔNACHT, p = 0.5413; Full Length vs. ΔLRR: p = 0.6035(O). n = 12 mice per group. Data are presented as mean ± standard deviation (SD). *p < 0.05, **p < 0.01, ***p < 0.001, n.s., non-significant.