Gene silencing effects of the CpG-Stat3 siRNA in DC2.4 cells were determined by quantitative real-time PCR analysis of the Stat3 mRNA (Fig. 2d ). Maximum effect on Stat3 silencing was observed at relatively high concentration of CpG-Stat3 siRNA (1 μM) in serum-containing cell culture medium. GpC-conjugated Stat3 siRNA, which binds but does not activate TLR9 36 , failed to silence Stat3, suggesting a possible requirement of TLR9 activation for the CpG-siRNA to be processed. Further experiments demonstrated that in TLR9 −/− myeloid cells and DCs, while cellular uptake of CpG-Stat3 siRNA was normal ( Supplementary Fig. 5 ), the gene silencing effect of CpG-siRNA was impaired (Fig 2e) . We also confirmed the gene silencing effects using electrophoretic mobility shift assays (EMSA) to detect Stat3 DNA-binding activity in DC2.4 cells, which was induced by IL-10 stimulation (Fig. 2f ). Note that, as indicated above, stimulation using CpG itself also resulted in Stat3 activation 25 , 38 (Fig. 2f) , thereby complicating the EMSA analysis for detection of Stat3 silencing. None-the-less, at higher concentrations, CpG-Stat3 siRNA diminished Stat3 DNA-binding activity relative to the conjugate scrambled RNA controls. To demonstrate the generality of this approach to gene silencing, we used TLR9-positive mouse A20 B cell lymphoma cells to test CpG-siRNA internalization ( Supplementary Fig.  6a ), and gene silencing, for which we tested a chimeric conjugate linking CpG1668 ODN with a Dicer substrate siRNA specific for firefly luciferase (Luc) in inhibiting luciferase activity in A20-Luc cells ( Supplementary Fig. 6 b) .