Fig. 11.

Role of GAS activation in p70-induced expression of iNOS in mouse BV-2 microglial cells. Cells were cotransfected with 0.2 μg of either pGAS-Luc (an STAT-dependent reporter construct) (A) or pISRE-Luc (an IRF-dependent reporter construct) (B) and 10 ng of pRL-TK. After 24 h of transfection, cells were stimulated with different concentrations of p40₂ and p70 for 6 h followed by analysis of firefly and Renilla luciferase activities. C: Cells were cotransfected with pGAS-Luc and pRL-TK. After 24 h of transfection, cells were treated with different concentrations of AG490 for 30 min followed by stimulation with 10 ng/mL p70. After 6 h of stimulation, luciferase activities were analyzed. D: Cells preincubated with different concentrations of AG490 for 30 min were stimulated with 10 ng/mL p70. After 6 h of stimulation, the expression of iNOS was monitored by RT-PCR. E: After 24 h of stimulation, concentration of nitrite was measured in supernatants. *P < 0.001 and **P < 0.05 vs. p70. F: Cells were co-transfected with siRNA constructs against IL-12Rβ1 or IL-12Rβ2 and pGAS-Luc. The pRL-TK was also used as transfection efficiency control. Twenty-four hours after transfection, cells were stimulated with 10 ng/mL p70 for 6 h followed by analysis of luciferase activities. Data are mean ± SD of three different experiments. *P < 0.001 vs. pSilencer-p70.