Decreased membrane expression of β-catenin in confluent FoxM1-deficient ECs. (A) Representative micrographs of immunofluorescent staining with anti–β-catenin antibody. At 65 h after transfection with either human FoxM1 scRNA, siRNA, or siRNA plus plasmid DNA expressing human β-catenin (siRNA⁺β-catenin, 1.5 µg/10⁶ cells), monolayers were fixed and immunostained with anti–β-catenin antibody to visualize β-catenin (red). Nuclei were counterstained with DAPI (blue). The experiment was performed three times with similar results. Bar, 20 µm. (B) Quantification of fluorescent intensity demonstrating reduced membrane expression of β-catenin in FoxM1-deficient HMVEC-L, which was restored by exogenous β-catenin expression. Accumulation of β-catenin at the membrane was quantified using 12-bit depth confocal z-series images. Maximum pixel values from different z sections were projected to a single plane using MetaMorph 7.1.0. Integrated fluorescence intensity of threshold projected images was calculated for β-catenin. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. **, P < 0.01 versus siRNA. (C) Western blot analysis demonstrating decreased β-catenin protein levels in the membrane fraction in confluent FoxM1-deficient HMVEC-L. At 65 h after transfection, confluent HMVEC-L monolayers were collected for cell fractionation. Each membrane fraction or cytosolic fraction (7.5 µg per lane) was loaded for detection of β-catenin with anti–β-catenin antibody. The experiment was performed three times with similar results.