(A) 293T cells and (B) 293T-TLR3 cells were cotransfected with IFN-β or ISRE promoter luciferase reporter (100 ng), Renilla luciferase internal control (pRL-TK), and empty vector or increasing concentrations of pcDNA3.1-HA-DHX29 (0, 50, and 100 ng). Transfected 293T and 293T-TLR3 cells were stimulated with intracellular (IC) HMW Poly(I:C) (5 μg/ml) and LMW Poly(I:C) (10 μg/ml), respectively. Fold changes in IFN-β-luciferase (Luc) and ISRE-Luc activities were determined. (C) IFN-β-Luc and ISRE-Luc activities in 293T cells stimulated with Poly(dA:dT) (5 μg/ml) (D) IFN-β-Luc and ISRE-Luc activities in 293T cells stimulated with IC LMW Poly(I:C) (5 μg/ml). (E and F) Real-time PCR of IFN-β (IFNB) mRNA (E) and immunoblot analysis (F) of TBK1 and IRF3 phosphorylation in empty vector- and pcDNA3.1-Flag-DHX29 (100 ng)-transfected 293T cells treated with IC HMW Poly(I:C), IC LMW Poly(I:C), or Poly(dA:dT). (G) Real-time PCR analysis of DHX29 mRNA expression in different human tissues by two different primer sets (Primer set #1 and Primer set #2). (H) Cell lysates (30 ug of total protein) of human lung cell lines MRC5, A549, H441, monocyte, dendritic cell (DC), T cell, B cell, mouse MEF, CD4-T cells, CD19-positive B cells, BMDM, BMDC, peritoneal macrophage (pMac), RAW 264.7, neutrophil were loaded on SDS-PAGE for western blotting analysis using anti-DHX29 and anti-β-actin antibodies. Data from (A-E) are plotted as the mean ± s.d. Results are representative of three independent experiments. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. NS, not significant.