Factor B synthesis and release after stimulation with DNA requires HMGBs but not RAGE A. Expression of HMGB1 in RAW264.7 cell clones stably transfected with siRNA against HMGBs (siHMGB), or control siRNA (Ctrl). (A) Clonal cell line (siHMGB-12) expressing relatively low levels of HMGB1 was selected for future experiments. (B.) Factor B mRNA expression in control or siHMGB-12 cells 6 h after stimulation with calf thymus DNA (10 μg/mL) complexed with Lipofectamine 2000. *p < 0.001 versus unstimulated cells containing control siRNA vector, #p < 0.001 versus cells containing control siRNA vector stimulated with DNA/lipofectamine. (C) Factor B mRNA expression in peritoneal macrophages from WT and RAGE-deficient mice after stimulation with calf thymus DNA (10 μg/mL) alone, or calf thymus DNA (10 μg/mL) complexed with Lipofectamine 2000 for 6 h. *p < 0.01 versus WT control, #p < 0.01 versus unstimulated RAGE-deficient cells. Experiments were performed in triplicate and repeated at least three times.