Fig. 6

TGF-ß2/TGFBR2 signaling regulates microglial homeostasis via CX3CR1. a Immunofluorescent staining of representative sections showing TGF-β2 and NG2 immunoreactivities in the corpus callosum of wild-type mice. Arrows indicate double-labeled cells. Scale bar, 50 μm. b Measurement of TGF-β2 protein levels was performed in the concentrated conditioned medium (CM) from primary NG2 glial cells using TGF-β2 ELISA (n = 6 for control medium, n = 7 for NG2 glia CM). Unpaired t test. **P < 0.01. c Pre-incubation of NG2 CM with antibody against TGF-ß2 markedly reduces the mRNA levels of CX3CR1 and increases IL-1β expression in primary cultured microglia. Unpaired two-tailed t test, (n = 8–10). ***P < 0.001 for CX3CR1. Nonparametric test with Mann–Whitney test, **P < 0.01 for IL-1β. All data are expressed as mean ± SEM. d qPCR analysis shows that the increases in the expression of Tmem119 and CX3CR1 in microglial cell line BV2 cells were abolished upon addition of LY2109761, a TGFBR1/2 blocker. One-way ANOVA with Newman–Keul’s post test (6–8 independent experiments). *P < 0.05, **P < 0.01, ****P < 0.001. e Immunoblotting analysis reveals that the elevations in the levels of phopho-Smad2 evoked by TGF-β2 are blocked by LY2109761 in BV2 cells. One-way ANOVA with Newman–Keul’s post test (n = 3 independent experiments). ****P < 0.001. f qPCR analysis shows that the decreases in the expression of CX3CR1 and Tmem119 in BV2 cells were induced upon transfection of TGFBR2 siRNA #1 or #2. One-way ANOVA with Newman–Keul’s post test (n = 4 independent experiments). ****P < 0.001. g Representative western blots from two separate experiments showing the decreases in the levels of phopho-Smad2 in both the cerebrocortex and striatum of mice with ablated NG2 glia. h, i Quantification of data shown in g. Two-way ANOVA with Bonferroni’s test (n = 3 mice per group). *P < 0.05, **P < 0.01