DHX29 KD markedly reduced TBK1 and IRF3 phosphorylation in response to intracellular HMW Poly(I:C) treatment, but not in response to LMW Poly(I:C) or Poly(dA:dT), in 293T cells (Fig 2A). Consistent with these results, DHX29 KD greatly reduced ISRE-Luc and IFN-β-Luc activities in 293T cells stimulated with intracellular HMW Poly(I:C), which could be rescued by ectopic expression of HA-DHX29 (Fig 2B). However, DHX29 KD had little or no effect on ISRE-Luc and IFN-β-Luc activities in 293T-TLR3 cells stimulated with LMW Poly(I:C) or 293T cells stimulated with intracellular LMW Poly(I:C) or Poly(dA:dT) (S2E–S2G Fig). Furthermore, we found that the expression of several endogenous genes, including IFN-β, IFIT1 and IFIT2, were reduced in cells transduced with DHX29-specific shRNAs in response to intracellular HMW Poly(I:C), but not intracellular LMW Poly(I:C) or poly(dAdT) (Fig 2C and S2H–S2J Fig), suggesting specific effects of DHX29 on HMW Poly(I:C)-induced type I IFN signaling.