Overexpression of S100A16 promoted EMT in MCF-7 cells. (A) Compared with control cells (MCF7-GFP), MCF7-S100A16 cells showed spindle-like, fibroblastic morphology (10×). (B, C) qRT-PCR (B) and western blot (C) analyses showed that epithelial markers E-cadherin and β-Catenin were significantly reduced in mRNA and protein levels in MCF7-S100A16 cells compared with MCF7-GFP cells, and mesenchymal markers Vimentin and N-cadherin were significantly up-regulated in MCF7-S100A16 cells (Bars, mean ± SD, P < 0.05). (D, E) Immunofluorescence staining was used to examine the location of epithelial and mesenchymal markers. After fixation, the celluar location of E-cadherin (red), β-catenin (red), Vimentin (red) and N-cadherin (red) were analyzed by confocal microscopy. Cell nuclei were stained with DAPI (4′, 6-diamidino-2-phenylindole, blue). Immunofluorescence staining showed that both E-cadherin (red) and β-catenin (red) resided in the cell membrane and intensity of fluorescence were reduced in MCF7-S100A16 cells compared with MCF7-GFP cells (D), whereas the mesenchymal markers Vimentin (red) and N-cadherin (red) were increased by S100A16 (E).