E-cadherin suppression in breast cancer stem cells (bCSCs) is associated with greater nuclear translocation of β-catenin and subsequent trans-activation of Slug. (A) β-catenin-associated E-cadherin was assayed by co-immunoprecipitation from cell lysates of MCF-7 and 2° mammospheres by using specific antibodies (left panel) or with normal human immunoglobulin G (IgG) as a negative control (right panel). To ensure comparable protein loading, 20% of supernatant from immunoprecipitation (IP) sample was subjected to determination of α-actin by Western blotting (WB). (B) WB was conducted to study the levels of total β-catenin and nuclear β-catenin in MCF-7 and 2° mammospheres for determining the nuclear translocation of β-catenin. (C) The relative nuclear expression of β-catenin in MCF-7 and 2° mammospheres was visualized by immunofluorescence. (D) WB was performed to study the expression levels of β-catenin target genes Cyclin-D1, c-Myc, Slug and Snail in MCF-7 cells and 2° mammospheres. (E) Protein and mRNA expression profiles of E-cadherin in 2° mammospheres of MCF-7 cells with or without transfection with Slug-short interfering RNA (siRNA) were determined by WB (right panel) and reverse transcription-polymerase chain reaction (RT-PCR) (left panel). The efficiency of transfection was assessed by evaluating the expression of Slug through WB (inset). (F, G) Graphical representation of relative cell adhesion, spreading, three-dimensional invasion, and transwell migration of MCF-7-derived 2° mammospheres with or without transfection with Slug siRNA. Transwell migration assay was also performed under similar experimental conditions in T47D-derived 2° mammospheres (G, right panel). α-Actin/histone H1/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.