Accumulation of cellular β- and γ-catenin reduces influenza A virus progeny. (A and B) A549 cells were transfected with the indicated plasmids (1 μg each but 2 μg per 6-well dish) for 24 h and subsequently infected with FPV (MOI = 0.01) for an additional 24 h. Transfection efficiency was verified by Western blotting. Equal protein loads were verified by β-actin immunoblotting (A). Virus propagation was analyzed by standard plaque titration assay (B). Representative images of three independent experiments are depicted. (C) A549 cells were stimulated with 20 mM LiCl or 100 mg/ml recombinant Wnt3a for 20 h and the induction of β-catenin signaling was verified by measuring the activity of the transfected TopFlash reporter gene. Luciferase activity of unstimulated cells was taken as unity. (D) A549 cells were prestimulated with 20 mM LiCl or 100 mg/ml Wnt3a for 3 h and 16 h, respectively, and infected with FPV (MOI = 0.01) for 24 h. Virus propagation was analyzed by standard plaque titration assay and the titer of unstimulated cells was arbitrarily set to 1. One representative experiment out of three is shown. (E and F) The endogenous β-catenin of SW480 cells was knocked down by specific siRNA. At 24 h post transfection, cells were infected with FPV (MOI = 0.001) for an additional 24 h, and viral titers (E) as well as knockdown efficiency (F) were determined. The β-actin immunoblot served as loading control. Protein scores of β- and γ-catenin presented in the right panel of (F) were estimated densitometrically as the relative intensity of the respective protein bands to the loading control. Mean values of control cells were taken as unity. (G and H) A549 cells were transfected with the indicated plasmids for 24 h and subsequently infected and analyzed as in (A and B).