Extended Data Fig. 3: TGFα and VEGF-B are regulated by AHR in highly purified astrocytes and microglia. | Nature

Extended Data Fig. 3: TGFα and VEGF-B are regulated by AHR in highly purified astrocytes and microglia.

From: Microglial control of astrocytes in response to microbial metabolites

Extended Data Fig. 3

a, b, Mouse microglia were activated with lipopolysaccharide (LPS) in the presence or absence of the AHR inhibitor CH223191. After 24 h, activation medium was removed and substituted with fresh medium after extensive washes. Then 48 h later, microglia conditioned medium (MCM) was collected and applied to cultures of primary astrocytes. a, Gene expression in microglia 24 h after activation in the presence or absence of CH223191. b, Gene expression in astrocytes after 24 h exposure to MCM. Data are representative of two independent experiments with three biological replicates. c, Representative FACS stainings for CD11b and CD45 in primary astrocyte and microglia cultures. Numbers indicate percentages in respective gate. Data are representative of three independent experiments. d, Representative FACS stainings for GFAP and GLAST in astrocyte cultures as in b. Data are representative of three independent experiments. e, f, qPCR analysis of mRNA expression in astrocyte and microglia cultures. n = 4 independent cultures. Data are representative of two independent experiments with four biological replicates. g, Effect of TGFα and VEGF-B on gene expression in primary astrocytes activated with TNF and IL-1β, determined by pPCR after 24 h. Data are representative of three independent experiments with three biological replicates. h, i, Primary mouse astrocytes were activated with TNF and IL-1β and treated with TGFα or VEGF-B. After 24 h later, culture medium was substituted by fresh medium after extensive washes. Then 48 h later, ACM was added to mouse neurons (h) and oligodendrocytes (i) in culture, and cytotoxicity was determined by quantifying lactate dehydrogenase (LDH) release after 24 h. n = 3 biological replicates. Data are representative of two independent experiments. j, CD11b+Ly6Chi monocyte migration assay performed using ACM from astrocytes activated in the presence of TGFα or VEGF-B. n = 4 biological replicates. Data are representative of two independent experiments. k, qPCR analysis of Nos2 expression in microglia co-cultured with astrocytes activated in the presence of TGFα or VEGF-B. n = 3 biological replicates. Data are representative of two independent experiments. Data in b, ek are mean ± s.e.m. P values were determined by two-sided Student’s t-test (b, e, f) or one-way ANOVA followed by Tukey’s post-hoc test (gk). 

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