Figure 2. Environmental chemicals boost astrocyte pathogenicity.
A) Pathway analysis of ToxCast-identified genes for Linuron and the unfolded protein response. B) Western blot and quantification for treated neonatal astrocytes. n=3 P-p65, n=3 P-IRE1α, n=4 XBP1. Kruskal-Wallis non-parametric ANOVA, uncorrected Dunn’s post-test. C) Western blot. n=4 per condition. Unpaired two-tailed t-test. D) qPCR. n=6 per condition. Unpaired two-tailed t-test. E) Thioflavin T (ThT) fluorescence in neonatal astrocytes treated for 24 hours. n=5–6 images per condition from N=3 cultures. One-way ANOVA, Dunnett post-test. F) (Left) Quantification of Xbp1 splicing. (Right-top) Schematic of Xbp1 splicing assay. (Right-bottom) Splicing assay gel. n=5–6 per condition at 4 hours; n=9 per condition at 24 hours except Linuron (n=3). One-way ANOVA, Bonferroni post-test. G) qPCR in neonatal astrocytes after 24h treatment. n=6–9 per condition, n=4 for Csf2 Linuron condition. One-way ANOVA, Bonferroni post-test, performed on ln-normalized data for Nos2, Il6, and Csf2. H) qPCR of Nos2 expression in neonatal primary mouse astrocytes. n=6 per condition, n=3 for GSK2656157 condition. One-way ANOVA, Holm-Sidak post-test. I) (Left) Predicted XBP1 and p65 binding sites in the murine Nos2 and Csf2 promoters. (Right) ChIP-qPCR of XBP1 and p65 recruitment to Nos2 and Csf2 promoters in neonatal primary mouse astrocytes. n=5 for p65 vehicle, n=2–3 for Linuron, n=6 otherwise. Kruskal-Wallis non-parametric ANOVA, uncorrected Dunn’s test (Csf2, XBP1 site 2) or one-sample t-test (otherwise). J) HEK293 luciferase reporter assay at the indicated hours post transfection (hpt). n=5–6 per group. One-way ANOVA, Dunnett post-test. See also Figure S1 and Table S2.