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. 2019 Oct 30;142(12):3806–3833. doi: 10.1093/brain/awz319

Figure 3.

Figure 3

The Wnt pathway is downregulated in pro-inflammatory microglia and modulates microglia activation in vitro. (A) Transcriptomic data reveal that the Wnt signalling pathway is strongly associated with MG/Mϕ activation in our model and (B) builds a cohesive network of multi-modal interactions between Wnt pathway genes in the MG/Mϕ co-expression network. (C) Validation of a selection of Wnt targets from the array data showing min to max box and whiskers plots of mRNA level presented as a fold-change relative to PBS group by RT-qPCR in CD11B+ MG/Mϕ from PBS or IL-1β-exposed mice. See also Supplementary Fig. 3. Two-way ANOVA, post hoc Bonferroni’s test, *P < 0.05, **P < 0.01 ***P < 0.001, n/group is indicated on the graph. (D) Scatter plot of β-catenin protein level from ELISA in CD11B+ MG/Mϕ from PBS or IL-1β-exposed mice at P3. Data are expressed as fold-change relative to PBS group (mean ± SEM, Student’s t-test, *P < 0.05, n = 9/group). (E) β-Catenin pathway inhibition, with XAV939, induced a pro-inflammatory like activation in primary microglia. Min to max box and whiskers plots of mRNA levels are presented as a fold-change relative to vehicle group (Student’s t-test, *P < 0.05, **P < 0.01, n = 12/group). (F) β-Catenin pathway activation induced by blocking Axin2 with siRNA (see also Supplementary Fig. 3I, ∼45% Axin2 mRNA decrease). (G) Inhibition of GSK3β, a β-catenin inhibitor, with CT99021 reduced primary microglia activation induced by IL-1β. Min to max box and whiskers plots of mRNA levels presented as a fold-change relative to vehicle group. One-way ANOVA with post hoc Newman-Keuls’s test. Effects of IL-1β on gene expression are shown with +P < 0.05, ++P < 0.01, +++P < 0.001; effects of Axin2 siRNA or CT99021 to alter the IL-1β effects are shown with *P < 0.05, **P < 0.01, ***P < 0.001, n = 6/group). (H) Effects of modulating Wnt with XAV939, Axin2 siRNA and CT99021 on phosphorylation of β-catenin (PSer45 β-catenin/β-catenin ratio) in primary microglia. Scatter plots, mean ± SEM, Mann-Whitney test *P < 0.05, n = 3/group.