Figure 9.

CSs synthesized by C4ST-1 controlled endocytosis of N-cadherin, cleavage of N-cadherin, β-catenin signaling, and cell invasive activity in BT-549 cells. A, after BT-549 and C4ST-1KO cells were treated with or without Pitstop2 or nystatin for 2 h, the cell-surface proteins were labeled with Ez-Link sulfo-NHS-Biotin (2nd to 4th, 6th to 8th, 10th to 12th, and 14th to 16th lanes), which was followed by a pulldown assay using StreptAvidin-agarose beads. The pulldown complex was analyzed by immunoblotting using anti-N-cadherin. The box-and-whisker graph at right indicates the cell-surface expression level of N-cadherin, normalized against the total expression level of N-cadherin (input), in BT-549 and C4ST-1KO cells (n = 3). Statistical significance was assessed using a Student's t test. B, surface N-cadherin expression on BT-549 cells (magenta), BT-549 cells treated with Nystatin (orange) or with Pitstop2 (green), and on C4ST-1KO cells (cyan) were compared by FACS analysis. The shaded area indicates cells treated without anti-N-cadherin antibody (negative control). C, disappearance rate of the full-length N-cadherin in BT-549 and C4ST-1KO cells during a cycloheximide chase in the presence or absence of an inhibitor of endocytosis, NH₄Cl, was analyzed by Western blotting. In the graph at right the presence or absence of NH₄Cl is indicated by closed and open circles, respectively. Two independent experiments were carried out for each data set. Error bars represent standard deviation (S.D.). D, amount of the C-terminal fragment, N-Cad(C), generated in BT-549 and C4ST-1KO cells in the presence or absence of 10 μm MG132 was analyzed by Western blotting. In the graph at right, the quantified expression level of N-Cad(C) was compared between BT-549 and C4ST-1 cells. Three independent experiments were carried out for each data set. E, panel a, activation level of β-catenin signaling in BT-549 and C4ST-1KO cells was estimated using the TCF reporter vector (n = 3). Panel b, gene expression level of MMP9 in BT-549 and C4ST-1KO cells was examined using real-time PCR (n = 3). F, panel a and b, invasive activity of BT-549 and C4ST-1KO cells was measured in a Matrigel invasion assay in the absence or presence of CS-E (n = 3).