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. Author manuscript; available in PMC: 2019 Jan 23.
Published in final edited form as: Nat Neurosci. 2018 Jul 23;21(8):1049–1060. doi: 10.1038/s41593-018-0192-3

Fig. 5. Selective effect of Eed inactivation on stMg and cbMg gene expression.

Fig. 5

(a) Schematic showing microglia-specific Eed deletion in adult mice. (b) H3K27me3 levels were quantified by Western blot analysis of isolated microglial nuclei (50,000 nuclei from n=3 mice/genotype) relative to total H3 (2 independent experiments). Representative blot (left, cropped to show the specific band); quantification (right). Ratio of intensities (ImageJ) from control, Cx3cr1CreErt2/+; Eef1a1LSL.eGFPL10a/+; Eedfl/+ (mean= 0.9245, SEM=0.04116), and mutant, Cx3cr1CreErt2/+(Litt) ; Eef1a1LSL.eGFPL10a/+; Eedfl/fl mice (mean=0.01006, SEM=0.00556). p < 0.0001, F=54.90, t4=22.02. Two-tailed unpaired t-test. (c) Quantification of the number of H3K27me3+ cells from control (microglia: mean= 96.50; non-microglia: mean=89.70) and mutant mice (microglia: mean=3.500; non-microglia: mean= 90.33). > 50 cells from n=2 mice/genotype. Bar graphs with individual data points show mean ± SEM. (d) H3K27me3 (red) in YFP/GFP+ microglia (green) using immunofluorescence of brain sections (DAPI: blue). Scale: 10 μm. Representative image is shown (2 independent experiments). Dotted circles: microglial nuclei. (e) Bar graph shows number of genes up-/down-regulated in Eed-deficient stMg at 3, 6, and 9 months by TRAP (DESeq2, n=2/region/genotype). Number of H3K27me3+ genes (red) is shown. (f) Principle Component Analysis (PCA) of stMg-/cbMg-TRAP-seq of 3mo/6mo/9mo Cx3cr1CreErt2/+;Eef1a1LSLeGFPL10a/+;Eedfl/fl and control, Cx3cr1CreErt2/+; Eef1a1LSLeGFPL10a/+; Eedfl/+ mice (n=2/genotype/age). (g,h) MA plots show gene expression changes (red: up; blue: down) caused by deletion of Eed in stMg (g) and cbMg (h) of 9mo mice (DESeq2, n=2/genotype). x-axis: log2 (mean expression); y-axis: log2 (fold change). Genes in green are equally expressed. Pie charts show the GO-based categories of up-regulated genes with selected genes named.