Anti-Wnt-1 antibody inhibited β-catenin/Tcf4 transcriptional activity. A). Tcf4 reporter assay of Tcf-dependent transcriptional activity in Huh7 and Hep40 cell lines. Huh7 and Hep40 cells were co-transfected with plasmid encoding β-galactosidase (a control for transfection efficiency) and either the pTOPFLASH or pFOPFLASH reporters. Cells were incubated with anti-Wnt-1 antibody or control IgG (2 μg/ml) and harvested after 48 hr to measure luciferase and β-galactosidase activities. Reporter gene activation is expressed in terms of relative light units (RLU) detected in pTOPFLASH or pFOPFLASH transfected cells and normalized for β-galactosidase activity. The results are expressed as mean ± SD (error bars). Experiments were performed in triplicates; P < 0.05. B). Anti-Wnt-1 antibody decreased nuclear β-catenin accumulation in Huh7 and Hep40 cells. Histone 3 was used as the loading control. C). The effect of anti-Wnt-1 antibody on the expression of β-catenin/Tcf4 target genes c-Myc, cyclin D1, and survivin. Huh7 and Hep40 cells were incubated for 48 hr with anti-Wnt-1 antibody (2 μg/ml) and c-Myc, cyclin D1, survivin and β-actin (loading control) levels were determined by Western blotting using specific antibodies.