In vitro anti-proliferative and apoptotic effects of anti-Wnt-1 antibody on human HCC cell lines. A). Cell viability assays based on cellular ATP content were used to determine the effect of anti-Wnt-1 antibody on three human HCC cell lines and normal hepatocytes from three donors following 72 hr of antibody treatment. Relative ATP activity is proportional to the number of viable cells. Three independent experiments were done, each in triplicates. B). Phase-contrast microscopic examination of the effect of different concentrations of anti-Wnt-1 antibody on HCC cell line Huh7 and normal hepatocytes Hu0910. C). Pre-treatment with Wnt-1 specific blocking peptide abolished the anti-proliferative effect of anti-Wnt-1 antibody in Huh7 and Hep40 cells. Results are presented as mean ± SD (error bars). D). Anti-Wnt-1 antibody induced apoptosis in Huh7 cells. Huh7 cells were treated with anti-Wnt-1 antibody (10 μg/ml) with or without blocking peptide pre-treatment. After 72 hr incubation, cells were washed, fixed, and stained with TUNEL and PI as described under Materials and Methods to detect for apoptotic cells. Fluorescence labeling was visualized and photographed at 100× magnification.