The augmented migration potential of breast cancer stem cells (bCSCs) results from the suppression of the epithelial-mesenchymal transition (EMT) marker, E-cadherin. (A) Immunohistological staining for E-cadherin (brown color for antibody staining and counterstained with hematoxylin) of breast tumor samples. (B) Protein and mRNA expression profiles of E-cadherin in MCF-7 cells, 1° and 2° mammospheres, was determined by Western blotting (WB) (upper panel) and reverse transcription-polymerase chain reaction (RT-PCR) (lower panel). (C) Expression of E-cadherin in MCF-7 cells and 2° mammospheres was visualized by immunofluorescence. (D) Graphical representation of relative cell adhesion (left panel) and spreading (right panel) of MCF-7-derived 2° mammospheres with or without transfection with E-cadherin-short hairpin RNA (shRNA). The efficiency of transfection was assessed by evaluating the expression of E-cadherin through WB (inset). (E) A similar experimental setup was scored for three-dimensional (3D) invasion (left panel) and transwell migration (right panel) assays. Transwell migration assay was performed under similar experimental conditions in T47D-derived 2° mammospheres (right panel). α-Actin/glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal loading control. Data are presented as mean ± standard error of mean or representative of three independent experiments.