Supplementary Figure 7: Confirmation of differentially expressed genes in stMg and cbMg at RNA and protein level.

(a,b) Microglia cells that are positive for the indicated RNAs in striatum and cerebellum of brain sections from 4mo Cx3cr1^GFP/+ mice were determined using a combination of immunohistochemistry for GFP (green) with RNA in situ hybridization (ISH, red). ISH: Mrc1 (a, first row), Axl (a, second row), Apoe (a, third row), Tfec (a, fourth row), Ecscr (b, first row), Gpr56 (b, second row). (DAPI: blue). Scale: 10 μm. Representative image (left) and quantification of percentage of microglia cells that are positive for the indicated RNA ISH (right). Mrc1: stMg: mean=1.852, cbMg: mean=20.81. Axl: stMg: mean=22.36, cbMg: mean=61.11. Tfec: stMg: mean=4.688, cbMg: mean=25.73. Apoe: stMg: mean=35.62, cbMg: mean=75.75. Ecscr: stMg: mean=86.84, cbMg:mean=9.723. Gpr56: stMg: mean=67.59, cbMg: mean=14.17. > 50 cells from n=2 mice. Dotted circles indicate microglial soma. (c) Brain sections from 4mo Cx3cr1^GFP/+ mice were tested for RNA in situ hybridization (ISH, red) to positive and negative controls provided by manufacturer (RNAScope. DAPI: blue). Scale: 10 μm. Dotted circles indicate cells positive for ISH signal. The experiment was repeated independently twice with similar results. (d) MHCII, CD74, AXL, and ApoE expression (red) in GFP+/P2RY12+/IBA1+ microglia (green) were determined using immunofluorescence analysis from striatum and cerebellum of brain sections from 4mo Cx3cr1^GFP/+ or wild-type mice (DAPI: blue). Scale: 20 μm. Representative image (left) and quantification of ratio protein area / microglia area (right) is shown. MHCII: stMg: mean=0.02166, cbMg: mean=0.3239; 8 images/region from n=2 mice. CD74: stMg: mean=0.023, cbMg: mean=0.16; 8 images/region from n=2 mice. AXL: stMg: mean=0.2550, cbMg: mean=0.7506; 10 images/region from n=2 mice. ApoE: stMg: mean=5.585, SEM=2.097; cbMg: mean=22.81, SEM=1.829; p=0.0035, F=1.315, t₄=6.189; 11 images/region from n=3 mice. A two-tailed paired t-test was performed for all quantifications. Bar graphs with individual data points show mean ± SEM. (e) Apoptosis of human Jurkat cells was induced with 1 μM Staurosporine for 3 hours, and induction of apoptosis was verified using FITC-Annexin V / propidium iodide kit. Quantification of necrotic (mean=0.4067, SEM=0.1225), late apoptotic (mean=5.877, SEM=0.3910), live (mean= 17.48, SEM=1.589), and early apoptotic (mean=76.23, SEM=1.492), p<0.0001, F=990.1. One-way ANOVA with Tukey’s Multiple Comparison post-hoc analysis to compare all pairs of columns (n=3 wells of Jurkat cells). ***p ≤ 0.001. (f) Representative flow cytometry histogram (related to Fig. 2d) overlay of stMg / cbMg isolated from 3/4mo Cx3cr1^GFP/+ mice illustrates that cbMg are more efficient in engulfment of pHrodo-labeled early apoptotic Jurkat cells. x-axis: pHrodo intensity. Histograms show cbMg / stMg (1) alone, (2) with early apoptotic cells, and (3) with early apoptotic cells and Cytochalasin D (phagocytosis inhibitor). The experiment was repeated independently 4 times with similar results.