Figure 3.

DNMT1-regulated CLDN6 expression inhibited tumor cell migration and invasion by blocking epithelial to mesenchymal transition (EMT). (A) Representative light microscope images of wound-healing assays for MCF-7 and SKBR-3 cells after treatment with or without SB431542 to evaluate their migration rate into the cell-free area (bar, 200 μm). (B) Matrigel invasion assay. Cells that invaded through the Matrigel were stained with Giemsa and counted. All results are presented as the average of cells counted in 10 fields per condition (bar, 50 μm). (C) RT-PCR; and (D) Western blotting analysis was used to determine the expression of EMT-related genes (SNAIL, E-cadherin, N-cadherin, and vimentin) in MCF-7 and SKBR-3 cells. Results of densitometric analysis of relative expression levels after normalization to loading control GAPDH or β-actin are presented. (E) Immunofluorescence analysis of the expression levels of EMT markers in MCF-7 and SKBR-3 cells. Representative immunofluorescence images (200×) generated using anti-E-cadherin, anti-N-cadherin, and CLDN6 primary and FITC-conjugated secondary antibodies in the breast cancer cells. Blue, cell nuclei were stained with 4,6-diamidino-2-phenylindole. Localization of E-cadherin, N-cadherin, and CLDN6 (red) at the cell–cell junctions is indicated by white arrows (bar, 20 μm). ** p < 0.01. Bars represent mean ± SE (n = 3).