FIGURE 7.

GnT-V regulates canonical Wnt/β-catenin signaling pathway. A, total RNA was pooled from four adjacent and adenoma tissues of GnT-V wild-type and knock-out Apc^min/+ mice, and expression of Wnt target genes (two left panels) and cytokeratin 20 (right panel) were detected by qRT-PCR. B, expression of Wnt target genes was detected by qRT-PCR using total RNA from control and GnT-V transfected LS180 (left) and GnT-V siRNA transfected HT-29 (right) cells. For each transcript the values were normalized to control (GAPDH) and expressed as the mean ± S.D. from three independent experiments. C, adjacent (control) and adenoma tissues were collected and used to detect protein expression by immunoblotting; Adj, adjacent; Tu, adenoma. D, GnT-V transfected colon cancer cell lines were used to detect protein expression by immunoblotting. C, control cells; G, GnT-V transfected cells. β-Actin was used as a loading control. E, c-myc expression and nuclear translocation of β-catenin were visualized by immunochemical staining of tumor tissues formed from injection of CCSC isolated from GnT-V transfected LS180 cells (middle panel) and adenomas of Apc^min/+ mice with different GnT-V genotypes (left panel); bar, 20 μm. Quantification of differences in nuclear β-catenin-positive cells (percentage of positive cells in six random fields of control or GnT-V transfected cells or per adenoma in Apc^min/+ mice with wild-type or GnT-V knock-out genotypes) (right panel). F, TOPFLASH/FOPFLASH analysis of Wnt activity of cultured colon cancer cell line with and without GnT-V transfection. The results are expressed as the mean ± S.D. of three independent experiments. *, p < 0.05. G, Aldefluor assays were performed using HT-29 cells treated with Wnt inhibitor (CCT036477, 20 μm, left panel) or LS180 cells with Wnt ligand (Wnt3a, 100 ng/ml, right panel) for 24 h, and the percentage of Aldefluor-positive cells was determined using similar gating criteria. Data are representative of two independent experiments.