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. 2023 Feb 20;3(3):275–296. doi: 10.1038/s43587-023-00368-3

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© The Author(s) 2023

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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Extended Data Fig. 8 — a, Volcano plot of the differentially expressed (DE) genes between microglia subcluster 4 and all other microglia subclusters. Gene names in red are marker genes for DAM. Dashed lines represent log2 fold change threshold of 0.4 and p-value threshold of 0.00001. b, Volcano plot of the DE genes between microglia subcluster 6 and all other microglia subclusters. Dashed lines represent log2 fold change threshold of 0.4 and p-value threshold of 0.00001. c, Volcano plot of the DE genes between microglia subcluster 8 and all other microglia subclusters. Dashed lines represent log2 fold change threshold of 0.4 and p-value threshold of 0.00001. d, Volcano plot of the DE genes in microglia subcluster 8 in PS19-fE4/Syn1-Cre mice versus PS19-fE4 mice. Dashed lines represent log2 fold change threshold of 0.4 and p-value threshold of 0.05. e, Dot-plot of the top 20 KEGG pathways significantly enriched for the DE genes of microglia subcluster 4 versus all other microglia subclusters (over-representation or enrichment analysis). f, Dot-plot of the top 20 KEGG pathways significantly enriched for the DE genes of microglia subcluster 6 versus all other microglia subclusters (over-representation or enrichment analysis). g, Dot-plot of the top 20 KEGG pathways significantly enriched for the DE genes of microglia subcluster 8 versus all other microglia subclusters (over-representation or enrichment analysis). h, Immunohistochemical staining for Iba1 and two markers (Tmsb4x and Ubb) of the disease-associated microglia subclusters 6 and 8 in the hippocampus (scale bar, 50 µm). i, Quantification of the number of Iba1+ microglia double-positive for Tmsb4x and Ubb in the hippocampus. Mice in h,i are 10-month-old. For data in a,b,c,d, the unadjusted p-values and log2 fold change values were generated from the gene-set enrichment analysis (see Methods for details) using the two-sided Wilcoxon Rank-Sum test. For data in e,f,g, the p-values are based on a hypergeometric test and are adjusted for multiple testing using the Benjamini-Hochberg method. The size of the dots is proportional to the number of genes in the given gene set. Data in i are n = 8 mice per genotype and represented as mean ± SEM, one-way ANOVA with Tukey’s post hoc multiple comparisons test.

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