IAV infection inhibits β-catenin-mediated transcriptional activation of LEF/TCF-dependent target genes. (A and B) A549 cells were transfected with the TopFlash reporter construct together with an empty vector or a plasmid coding for β-catenin. At 24 h post transfection, the cells were infected with FPV (MOI = 5) or stimulated (via transfection) for an additional 8 h with 500 ng of cellular or viral RNA or left untreated. Subsequently, the promoter activity was measured. The luciferase activity of mock-infected or unstimulated but empty vector-transfected cells was taken as unity. (C) A schedule of signaling cascades activated by IAV RNA is depicted (adapted from [10, 55, 56]). (D and E) HEK293 cells were transfected with the TopFlash reporter gene construct together with β-catenin and plasmids coding for the indicated proteins. At 30 h post transfection, the promoter activity was measured (D). The luciferase activity of β-catenin and empty vector-transfected cells was arbitrarily taken as unity. Overexpression of the recombinant proteins was analyzed by Western blotting. The β-actin immunoblots always served as loading controls (E). (F) A549 cells transfected with the TopFlash reporter plasmid together with β-catenin were stimulated with 5 mM of BAY inhibitor or DMSO as control. At 16 h post stimulation, cells were transfected with 500 ng cellular or viral RNA for an additional 8 h and the promoter activity was measured. The luciferase activity of cells stimulated with cellular RNA was always taken as unity. (G and H) Vero cells were transfected with reporter gene plasmids containing either the IRF3 responsive elements of the IFN-β enhanceosome (G) or the ISRE motif (H) along with plasmids coding for proteins indicated in column legends. The promoter activity was measured 30 h post transfection. Luciferase activity of cells transfected with any vector control was arbitrarily taken as unity.