Fig. 1
Tamoxifen treatment reduces the levels of Stat3 and p-Stat3 in reactive astrocytes isolated from prion-infected mice. Primary astrocytes were treated with TAM or mock solution for 72 h and analyzed. A, B. Upper panels: immunofluorescence microscopy images of 22L-Cre+/−astrocytes co-immunostained for GFAP and Stat3 (A) or p-Stat3 (B) along with DAPI. Lower panels: quantification of integrated fluorescence intensity for Stat3 (A) and p-Stat3 (B), and morphometric analyses of cell area, perimeter and process number in 22L-Cre+/− astrocytes. C, D. Upper panel: immunofluorescence microscopy images of 22L-Cre−/− astrocytes co-immunostained for GFAP and Stat3 (C) or p-Stat3 (D) along with DAPI. Lower panels: quantification of integrated fluorescence intensity for Stat3 (C) or p-Stat3 (D), and morphometric analyses of cell area, perimeter and process number in 22L-Cre−/− astrocytes. Images are representatives of three cultures originating from independent animals per experimental group. For Stat3 or p-Stat3 intensity, n = 10–12 random fields with 6–7 cells per field of view from N = 3 independent cultures, each prepared from an individual animal, per group. For morphology analysis, n = 80–100 cells from N = 3 independent cultures, each prepared from an individual animal, per group. In A-D SuperPlots: colors represent independent experiments; dots represent in individual fields of view or cells; 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. E. Analysis of expression of Stat3 in 22L-Cre+/− and 22L-Cre−/− astrocytes normalized by the expression levels in mock-treated 22L-Cre+/− or 22L-Cre−/− astrocytes using qRT-PCR. F, G. Representative Western blots and densitometric analysis of Stat3 (F) and p-Stat3 (G) expression in 22L-Cre+/− and 22L-Cre−/− astrocytes normalized per expression of β-actin. For E-G, N = 3 independent cultures, each prepared from an individual animal, per group. For A-G, data represent means ± SE, ***p < 0.001, **p < 0.01, and ‘ns’ non-significant by two-tailed, unpaired t-test, n = 3 independent experiments. Scale bar = 50 μm