Figure 5

Factor B upregulation in macrophages after stimulation with DNA is independent of AIM2, DAI and TLR9, but dependent on MyD88. (A) Factor B mRNA expression in WT and AIM2-deficient mouse peritoneal macrophages 6 h after stimulation with calf thymus DNA (10 μg/mL) alone or calf thymus DNA (10 μg/mL) complexed with Lipofectamine 2000. *p < 0.001 versus WT control, ^#p < 0.001 versus unstimulated AIM2-deficient cells. (B) Expression of factor B and DAI in cell lysates of RAW264.7 macrophages pretreated with either control (CRT) or DAI-targeting siRNA and then stimulated for 24 h with DNA alone (10 μg/mL), Lipofectamine 2000 alone or DNA (10 μg/mL) complexed with Lipofectatmine 2000 as indicated. (C) Expression of factor B mRNA in peritoneal macrophages from WT and TLR9-deficient mice stimulated with calf thymus DNA (10 μg/mL) alone or calf thymus DNA (10 μg/mL) complexed with Lipofectamine 2000 for 6 h. *p < 0.001 versus WT control, ^#p < 0.001 versus unstimulated TLR9-deficient cells. (D) Factor B mRNA expression in peritoneal macrophages from WT and MyD88-deficient mice 6 h after stimulation with calf thymus DNA (10 μg/mL) complexed with Lipofectamine 2000. *p < 0.001 versus WT control, ^#p < 0.001 versus stimulated WT cells. (E) Factor B release into cell culture supernatants of peritoneal macrophages from WT and MyD88-deficient mice stimulated with calf thymus DNA (10 μg/mL) complexed with Lipofectamine 2000 for 6 h. (F) Factor B mRNA expression in cell culture supernatants from RAW264.7 macrophages pretreated with MyD88-inhibitor peptide or control peptide, then stimulated with DNA alone (10 μg/mL), Lipofectamine 2000 alone or with DNA (10 μg/mL) complexed with Lipofectatmine 2000 for 6 h as indicated. Experiments were performed in triplicate and repeated at least three times.