Fig. 5
Stat3-mediated astrocyte reactivity is induced by microglia-secreted factors. Astrocyte were cultured in the presence of 22L MCM and TAM or mock solution for 72 h and analyzed A. Left panels: immunofluorescence microscopy images of Cre+/− astrocytes co-immunostained for GFAP and Stat3 along with DAPI. Cre+/− astrocytes cultured in the absence of 22L MCM are presented as reference. Right panels: quantification of integrated fluorescence intensity of Stat3, and morphometric analyses of cell area, perimeter and process number of astrocytes. B. Representative Western blot and densitometric analysis of Stat3 expression normalized per expression of β-actin. C. Analysis of expression of genes associated with astrocyte reactivity in Cre+/− astrocytes cultured in the presence of 22L MCM using qRT-PCR. D. Left panels: immunofluorescence microscopy images of Cre+/− astrocytes co-immunostained for GFAP and p-Stat3 along with DAPI. Right panels: quantification of integrated fluorescence intensity of p-Stat3 and morphometric analyses of cell area, perimeter and process number. E. Representative Western blots and densitometric analysis of p-Stat3 expression normalized per expression of β-actin. In A and D, images are representatives of N = 3 cultures originating from individual animals; for Stat3 and p-Stat3intensity, n = 10 random fields with 5–6 cells per field from N = 3 independent cultures originating from individual animals; for morphology analysis, n = 70–80 cells from N = 3 independent cultures, originating from individual animals. In 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. In B, C and E, N = 3 independent cultures, originating from individual animals. In A-E, data represent means ± SE, **p < 0.01, *p < 0.05, ‘ns’ is non-significant by one-way ANOVA with Bonferroni multiple comparisons test (in A and D) and by two-tailed, unpaired t-test (in B, C and E). Scale bars = 50 µm