Fig. 2

Stat3 deletion mitigates the reactive phenotype of astrocytes isolated from prion-infected mice. Primary astrocytes were treated with TAM or mock solution for 72 h and analyzed. A, B. Left panels: analysis of GFAP (A) and C3 (B) gene expression in 22L-Cre^+/− and 22L-Cre^−/− astrocytes using qRT-PCR. Right panels: representative Western blots and densitometric analysis of GFAP (A) and C3 (B) expression normalized per expression of β-actin in 22L-Cre^+/− and 22L-Cre^−/− astrocytes. C, D. Analysis of expression of genes associated with astrocyte reactivity in 22L-Cre^+/− (C) and 22L-Cre^−/− (D) astrocytes. The expression levels in TAM-treated astrocytes were normalized relative to those in mock-treated astrocytes. In A-D, N = 3 independent cultures, each prepared from an individual animal, per group. Data represent means ± SE, ***p < 0.001, **p < 0.01, *p < 0.05 and ‘ns’ is non-significant by two-tailed, unpaired t-test. E, F. Left panels: immunofluorescence microscopy images of 22L-Cre^+/− (E) and 22L-Cre^−/−(F) astrocytes co-immunostained for GFAP and C3 along with DAPI. Right panels: quantification of the C3-positive area within GFAP-positive 22L-Cre^+/− (E) and 22L-Cre^−/−(F) astrocytes. Images are representatives of three cultures originating from independent animals. n = 13 random fields with 8–10 cells per field of view from N = 3 independent cultures, each prepared from an individual animal, per group. In E and F SuperPlots: colors represent independent experiments; dots represent in individual fields of view; average values for each experiment are shown as large circles; statistical analyses were performed based on the number of independent experiments; black lines mark means, ***p < 0.001, ‘ns’ is non-significant by two-tailed, unpaired t-test. Scale bar = 50 µm