Fig 1: CD11c+ DCs enhance IFN-a production of CD11c- cells via DC-SIGN.(A-D) mRNA expression of monocyte-derived DCs stimulated with EC-DNA (A), control ODN, ODN-2216 (B,D), or R837 (C) after treatment with control, TLR7, TLR9 (A-C), or IRF7 siRNA (D) was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. (E) Immunoblot of monocyte-derived DCs or Raji cells whole cell lysate for TLR9. ß-actin was used as loading control. (F) Analysis of TLR9 expression in monocyte-derived DC culture by flow cytometry. Number adjacent to gates indicates percentage of gated cells. (G) mRNA expression of sorted CD11c+ DCs and CD11c- cells stimulated with control ODN or ODN-2216 was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with ODN-2216 of CD11c- cells. (H) mRNA expression of sorted CD11c+ DCs and CD11c- cells stimulated with control ODN or ODN-2216 after treatment with control or DC-SIGN siRNA. Sorted cells were stimulated for 1h, washed and combined for 5h before mRNA expression was measured by real-time PCR, normalized to GAPDH and set as 1 in samples with control siRNA. Cells were stimulated with 10 µg/ml DNA or 5µM ODN in all experiments. Data are collated (mean ± s.d.) of four (A,B,G), three (H) or two (C,D) independent experiments with different donors or are representative of four (F) or two (E) independent experiments with different donors. *P<0.05, **P<0.01 (student’s t-test). EC-DNA: E. coli DNA.