Figure 5

OCT4 -transduced breast cells (OTBCs) exhibit an epithelial-to-mesenchymal transition (EMT) gene signature. (a) Microarray expression analysis of genes selected across parental (p48 and p86), OTBC lines (OTBCs48-L1 and L2, OTBCs86-L1, L4, and L6), and a subcutaneous tumor derived from OTBCs86-L1 (tumor 86-L1). Array trees were derived by unsupervised hierarchical clustering using markers of EMT, basal, luminal stem cell, cancer stem cell, and epithelial differentiation. Each colored square on the upper right represents the relative mean transcript abundance (in log₂ space); highest expression is red, average expression is black, and lowest expression is green. (b) Western blot detection of molecular markers in the parental lines (p86 and p48), OTBCs86-L1 through L6, and OTBCs48-L1. The following markers were analyzed: OCT4, NESTIN, CDH1, Maspin, vimentin, and N-CAM. Tubulin was used as a loading control. Results are representative examples of three independent experiments. (c) Detection of EMT transcription factor expression by quantitative real-time polymerase chain reaction (qRT-PCR). Gene expression levels were normalized to those of the parental cell lines (p86 and p48). Control cell lines used for gene expression analyses include human dermal fibroblasts and the human teratoma cell line NT2. (d) MicroRNAs (miRNAs) known to promote epithelial differentiation are detected by qRT-PCR. Levels of miRNA expression in OTBCs of miR-141, miR-200a, miR-200b, miR-200c, and miR-205 were normalized to those of the parental lines (p86 and p48). Analysis was performed by using miR-U6 as an internal control. Bar graphs represent the mean ± standard deviation of three independent experiments. CDH1, E-cadherin; N-CAM, neural cell adhesion molecule.