Activation of astrocytes co-cultured with mast cells. Astrocytes (3 × 106 cells) and mast cells (1 × 106 cells) were co-cultured in a 3:1 ratio. The anti-CD40 antibody (300 ng/ml), Jak inhibitor (10 μM AG490), 8-oxo-dG (300 μg/ml) or 2-APB (100 μM) was pretreated in astrocytes 1 h, 5, 10 and 10 min, respectively, before co-culture, and CD40 siRNA was transfected as described in "Methods". Fluro-3 AM (5.0 μM) was added to co-cultured-U87 cells or -primary astrocytes and incubated for 30 min. The [Ca2+]i level in U87 cells or in primary astrocytes was analyzed by confocal laser scanning microscopy. Rho-family GTPases or expressions of cytokine mRNA were determined in protein extracts and nuclear extracts by GST pull-down assay and RT-PCR, respectively. (A, B) Time course of [Ca2+]i level in co-cultured-U87 cells or -primary astrocytes, respectively, and in anti-CD40 antibody pretreatment. (C) The [Ca2+]i level after pretreatment with anti-CD40 antibody, CD40 siRNA or other inhibitors. (D) The activation of Rho-family GTPases by various inhibitors. (E) Expressions of cytokine mRNA caused by various inhibitors. U87 cells, U87 cell culture alone; BAC, primary astrocyte culture alone; Co-culture, U87 cells co-cultured with HMC-1 cells or BAC with BMMCs; Anti-CD40 Ab, anti-CD40 antibody pretreatment; CD40 siRNA, CD40 siRNA transfected-U87 cells co-cultured with HMC-1 cells; AG490, Jak inhibitor; 8-oxo-dG, 8-hydroxydeoxyguanosine. *, The numbers below bands are mean values and Figure 1A, B and C are mean ± SEM obtained from four independent experiments (n = 4) as the ratio bad density of each group versus that of total protein or GAPDH using densitometry analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus U87 cell or primary astrocyte culture alone. ++, P < 0.01; +++, P < 0.001 versus co-cultured-U87 cells or -primary astrocytes.