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. 2022 Apr 12;8:34. doi: 10.1038/s41421-022-00377-3

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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. 3 — a Differential expression volcano plot of pseudobulk scRNA-Seq analysis. The S-phase microglia from day 1 after nerve injury displayed different gene expression profile from that of the S-phase microglia 2 days after nerve injury in Cx3cr1-creER-yfp/+; +/+ animals. b, c qRT-PCR of FACS-sorted microglia from lumbar spinal cord of Cx3cr1-creER-yfp/+; +/+ animals (b) and of RiboTag enriched microglia mRNA from lumbar spinal cord (c). Tnfaip3 was downregulated in the microglia of ipsilateral lumbar cord 1 and 2 days after nerve injury. d Quantification of proliferating Ki67 (+) microglia by flow cytometry. Naive uninjured Cx3cr1-creER-yfp/+; Tnfaip3 fl/fl A20 cKO animals had significantly more baseline microglia proliferation than those of naive control Cx3cr1-creER-yfp/+; +/+ animals. e scRNA-Seq PCA scatter plot of naive Cx3cr1-creER-yfp/+; Tnfaip3 fl/fl A20 cKO microglia. The distributions of G1, S, and G2/M phase cells were similar to that of Cx3cr1-creER-yfp/+; +/+ control animals 3 days after nerve injury as shown in Fig. 1d. f–h scRNA-Seq PCA scatter plot of Cx3cr1-creER-yfp/+; Tnfaip3 fl/fl A20 cKO microglia 1 day (f), 2 days (g), and 3 days (h) after nerve injury. The distributions of G1, S, and G2/M-phase cells were similar to that of naive Cx3cr1-creER-yfp/+; Tnfaip3 fl/fl A20 cKO animals (e). Analyses are two-way ANOVA with Sidak’s multiple comparison test (b, c) and unpaired two-tailed t-test (d), n = 4–6, means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.