Glia up-regulate galectin-9 following in vitro stimulation with poly(I:C). (A) Fold change in galectin-9 mRNA following stimulation of rat mixed glia with vehicle control (PBS) or poly(I:C) (50 μg/ml) over time. Results are combined means ± SE from two independent experiments. (B-E) Mouse mixed glia from Lgals9:EGFP mice (n = 4) stimulated with poly(I:C) (50 μg/ml). Time-dependent increase in both galectin-9 promoter activation (B) and protein production (C) following stimulation. Galectin-9 localizes to both Iba-1+ microglia (D) and GFAP+ astrocytes (E). (F) Relative Lgals9 expression in astrocyte (left) or microglia (right) monocultures following treatment with either recombinant TNF (5 ng/ml) or poly(I:C) (50 μg/ml). Results are means ± SE of duplicate samples and are representative of three independent experiments. (G) (H) Conditioned media from poly(I:C)-activated microglia is sufficient to induce Lgals9 expression in rat astrocyte cultures. Media obtained from microglia treated with vehicle (PBS) or poly(I:C) (50 μg/ml) for 24 hours was used to treat astrocytes for 7 hours and Lgals9 up-regulation determined by RT-PCR (left). Microglial conditioned media was heated to 100°C before adding to astrocytes (right). Results are representative of two to four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.