Extended Data Fig. 6: Regulation and transcriptional effects of TGFα and VEGF-B during EAE. | Nature

Extended Data Fig. 6: Regulation and transcriptional effects of TGFα and VEGF-B during EAE.

From: Microglial control of astrocytes in response to microbial metabolites

Extended Data Fig. 6

a, b, NanoString analysis of mRNA expression in astrocytes from EAE mice injected with pCD11b-shVegfb or pCD11b-shTgfa (a) and pGFAP-shFlt1 or pGFAP-shErbb1 (b; see also Fig. 2k, l). Fold change in relative expression relative to control as determined by log2(shKD/shControl). shKD, shRNA knockdown. Representative of two independent experiments with pooled RNA isolated from n = 3 mice per group. c, Principal component analysis of gene expression in astrocytes isolated as in a and b. Representative of two independent experiments with pooled RNA isolated from n = 3 mice per group. d, Ingenuity pathway analysis of significantly regulated pathways from astrocytes as in a and b. Representative of two independent experiments with pooled RNA isolated from n = 3 mice per group. e, Left, representative flow cytometry plots depicting NF-κB p65 phosphorylation in wild-type astrocytes stimulated for 15 min with vehicle (top) or TNF or IL-1β (bottom) in the presence of TGFα, VEGF-B, or their combination. Numbers indicate percentage of FITC+ cells. Bar graphs depict quantification of FITC+ cells. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with four biological replicates. f, Primary mouse astrocytes were exposed to VEGF-B or vehicle and pharmacological blocker of NF-κB activation. RNA was obtained after 18 h and subjected to qPCR analyses for the indicated genes. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with three biological replicates. g, Primary mouse astrocytes were activated with TNF or IL-1β in the presence of VEGF-B or vehicle, and a pharmacological blocker of NF-κB activation. RNA was obtained after 18 h and subjected to qPCR analyses for the indicated genes. Data are mean ± s.e.m. and P values were determined by one way ANOVA followed by Tukey’s post-hoc test. Representative of two independent experiments with n = 3 biological replicates. 

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