The ISG promoter activity is triggered by β- and γ-catenin. (A) A549 cells were transfected with β-catenin and LEF1 for 30 h, and the mRNA level of the type I and type III IFN-dependent MX1 gene was measured by qRT-PCR. The mRNA amount of empty vector-transfected cells was taken as unity. (B and C) Vero cells transfected for 24 h with indicated plasmids were infected with vesicular stomatitis virus (VSV) (MOI = 0.0001) for an additional 24 h. Subsequently, the overexpression of β-catenin was confirmed by immunoblotting of corresponding RIPA lysates (B) and the propagation of VSV by standard plaque titration assay (C). (D and E) Vero cells were co-transfected with the ISRE luciferase reporter gene and plasmids coding for proteins indicated in column legends. After 24 hours, Vero cells were left unstimulated or treated with 100 U/ml IFN-β for 8 h. The y-axis represents the relative reporter gene activity with luciferase activity of unstimulated, empty vector-transfected cells being set to one. (F) Vero cells were transfected with the ISRE luciferase reporter gene, and its activity in β-catenin- and LEF1-overexpressing cells was measured in the presence or absence of co-transfected p300. The luciferase activity of β-catenin and LEF1-transfected cells was arbitrarily taken as unity. (G) A549 cells were transfected with empty vector or plasmids coding for β-catenin and LEF1 for 30 h, and the interaction of cellular proteins with the DNA was analyzed by ChIP assays using specific antibodies to IRF3 or β-catenin. The co-immunoprecipitated DNA was amplified by qRT-PCR using specific primers for the promoter region of the MX1 gene and is given as the n-fold amount to the IgG control. Representative values from one of three repeated experiments are depicted.