Figure 4. Genomic control of disease-promoting astrocyte activities by IRE1α-XBP1 signaling.
A) qPCR in sorted adult astrocytes from naïve and EAE mice. n=3 per group. Unpaired two-tailed t-test per gene. B) qPCR of Nos2 expression (left) or XBP1 binding to the Nos2 promoter by ChIP (right) in sorted adult astrocytes from naïve and EAE mice. n=3 naïve, n=8 EAE (qPCR) or n=2 (ChIP) per group. Unpaired two-tailed t-test on data ln-normalized (qPCR) or not (ChIP). C) Schematic of shRNA lentiviral vector and EAE development in transduced mice. n=13 shScrmbl (left), n=13 shXbp1, n=10 shScrmbl (right), n=9 shErn1. Two-way repeated measures ANOVA, Holm-Sidak post-test. LTR=long terminal repeats, GfaABC1D=Gfap promoter, EGFP=enhanced green fluorescent protein, pA=polyadenylation signal, WPRE=woodchuck hepatitis virus post-transcriptional regulatory element. D) qPCR of Nos2 expression in EAE astrocytes. n=9 per condition. Unpaired non-parametric Kolmogorov-Smirnov t-test. E) RNA-seq analysis of astrocytes from EAE mice. Columns are n=4 biological replicates. Genes with p<0.05 are shown. F) Pathway analysis of astrocyte signaling in Xbp1 knockdown. G) Gene set enrichment analysis for astrocyte RNA-seq. H) ATAC-seq peaks in astrocytes. n=4 shScrmbl, n=3 shXbp1. Scale bars show read density. Transcriptional schematics shown above plots. I) EAE scores. n=7 Vehicle, n=16 sgScrmbl, n=9 sgXbp1, n=10 sgSigmar1. Two-way repeated measures ANOVA, Holm-Sidak post-test. U6=human U6 promoter, sgRNA=single guide RNA, 2A=self-cleaving peptide. J-K) Astrocyte Nanostring analyses from I. RNA pooled from n=4 mice (sgScrmbl) or n=7 mice (sgXbp1) in J, n=5 mice per group in K. See also Figures S2–S5 and Tables S3–S6.