Adenoviral overexpression of EpCAM in HMECs inhibits cell proliferation and migration. HMECs (n = 3) were adenovirally transfected to overexpress EpCAM and GFP or GFP alone. A multiplicity of infection of 100 viruses/cell was used for all experiments. Overexpression of EpCAM was confirmed 48 h after transfection by Immunofluorescence (A). EpCAM was expressed on cell surface and cytoplasm (Phycoerythrin, red signal). Nuclei were counterstained with DAPI (blue signal, magnification 400×, bars indicate 25 μm). EpCAM overexpression was analyzed by real time PCR using GAPDH as housekeeping gene for normalization and GFP transfected cells as controls (B). As expected, overexpression resulted in a more than hundred-fold induction of EpCAM gene expression even 5 days after transfection. Protein expression was confirmed by Western Blot analysis (C). In comparison to control cells EpCAM was overexpressed as glycosylated isoform in proliferating cells and primarily as not glycosylated isoform in growth arrested HMECs. EpCAM glycosylation has been analyzed by enzymatic deglycosylation experiments with PNGaseF and subsequent Western Blot analysis. In all samples we observed a reduction of the 40-42 kDa glycosylated isoforms to the 35 kDa not glycosylated EpCAM isoform (D). Cell proliferation was analyzed in real time by the use the xCelligence system. EpCAM overexpression significantly inhibited cell proliferation (E). Western Blot analysis of cell cycle inhibition 48 h after EpCAM overexpression; p53 and p27KIP1 proteins were upregulated in EpCAM overexpressing cells. (F). Cell migration was monitored by xCelligence CIM plate system after adenoviral transfection of EpCAM or GFP (G). EpCAM overexpression also inhibited cell migration; stars indicate p values < 0.05.