C8G induction in astrocytes under neuroinflammation. (A) Immunofluorescence analysis of C8G in the hippocampus. Mice injected with LPS (5 mg/kg) intraperitoneally (i.p.). Scale bar = 25 μm. Right: The number of GFAP-positive cells and co-localized cells per unit area in the hippocampus (n = 6) as well as averages of Pearson’s coefficients (n = 6). (B) Time course of C8G protein expression in the inflamed brain. C57BL/6 mice (n = 3) injected with LPS (5 mg/kg) i.p. (C) Comparison of mRNA expression of C8G, pro-inflammatory cytokines, and lipocalin-2 (Lcn2) in the LPS-injected brain of mice (n = 3). (D) C8G expression in different cell types of the CNS. Microglia, astrocytes, and bEnd.3 mouse brain endothelial cells (n = 3 each) were incubated with LPS (1 μg/ml) alone or with L/I [LPS (1 μg/ml) and IFN-γ (50 units/ml)] as indicated. Primary cultured hippocampal neurons were incubated with conditioned medium of mixed glial cells stimulated with L/I. After a 6 h treatment, C8G level was measured with traditional RT-PCR. (E) C8G induction by inflammatory stimuli in mouse primary cultured astrocytes (n = 3). Cells were incubated with L/I, recombinant mouse TNF-α (10 ng/ml), IL-1β (10 ng/ml), IL-6 (10 ng/ml), IFN-γ (50 units/ml), IL-10 (10 ng/ml), IL-4 (10 ng/ml), or IL-13 (10 ng/ml) for 6 h. Mouse liver was used as a positive control. (F) C8G mRNA induction by inflammatory stimuli in human astrocytes (n = 3). Astrocytes were incubated with recombinant human TNF-α, IL-1β, IL-6, IFN-γ, IL-10, IL-4, or IL-13 protein or their combination (Mix: TNF-α + IL-1β + IFN-γ) with the same concentration as mouse astrocytes for 6 h. HepG2 cells were used as a positive control. The assay (n = 3; done in triplicate) was repeated at least two times (inter-assay coefficients of variation were < 20%). Data are mean ± SEM. *P < 0.05 versus control (Con). Unpaired non-parametric test in A (Mann-Whitney) and D (Welch’s t-test); Dunnett's multiple comparison post hoc tests after one-way ANOVA in B, C, E and F.