Effect of TLR3/RIG-I activation on IFN regulatory factors (IRFs). (A and B) End1/E6E7 cells, transfected with or without poly I:C (A) or 5′ppp dsRNA (B) at indicated concentrations for 24 h, were subjected to western blot assay using antibodies to IRF3, IRF7 or GAPDH. GAPDH was used as the loading control. (C and D) End1/E6E7 cells were transfected with or without poly I:C (C) or 5′ppp dsRNA (D) at indicated concentrations for 12 h. (E and F) shRNA-mediated knockdown of IRF3 and/or IRF7 impaired IFN-λ expression. End1/E6E7 cells were transfected with or without control vector, IRF3 shRNA or IRF7 shRNA for 48 h. Cells were then transfected with or without poly I:C (E) or 5′ppp dsRNA (F) for additional 12 h. Total cellular RNA extracted from End1/E6E7 cells was subjected to the real-time RT–PCR detection for the mRNA levels of IRF3, IRF7 and IFN-λ. The data are expressed as mRNA levels relative (-fold) to control (without poly I:C or 5′ppp dsRNA stimulation, which is defined as 1). The results are mean ± SD of triplicate cultures, representative of three experiments (*P < 0.05, **P < 0.01).