(A) Immunoblot analysis showing DHX29 expression in THP-1 cells stimulated with intracellular (IC) HMW Poly(I:C) (A, upper) or EMCV (A, lower) at the indicated time points. β-actin was used as a loading control. (B) Real-time PCR analysis of IFNB and DHX29 mRNA levels in THP-1 cells stimulated with IC HMW Poly(I:C). (C) Real-time PCR analysis of the KD efficiency of DHX29-specific shRNAs. Scrambled (Scr) siRNA was used as a control. (D) 293T cells were cotransfected with HA-vector or HA-DHX29 and Scr shRNA or DHX29 shRNA. Cell viability was determined at various time points post-transfection using bromophenol blue. (E) ISRE-luciferase (Luc) (left panel) and IFN-β-Luc activities (right panel) in Scr shRNA- and DHX29 shRNA-transfected 293T-TLR3 cells stimulated exogenously with naked Poly(I:C). (F and G) ISRE-Luc (left panel) and IFN-β-Luc (right panel) activities in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with (F) IC LMW Poly(I:C) and (G) IC Poly(dA:dT). ISRE-Luc and IFN-β-Luc activities are expressed as the fold increase relative to the control. (H-J) Real-time PCR analysis of IFIT1, IFIT2, and CCL5 mRNA levels in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with IC HMW Poly(I:C). Data from (A-J) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to Fig 2 in the main text.