EpCAM overexpression leads to upregulation of c-myc and increased cell proliferation in immortalized MCF10A human breast epithelial cells. (A) Flow cytometric analysis of EpCAM expression in adenovirally transfected MCF10A cells. In comparison to GFP transfected controls, only EpCAM transfected cells show the immunoreactive protein on the cell membrane. (B) Overexpression of EpCAM results in a significant increase in cell proliferation under serum-reduced conditions. (C) Real time PCR analysis of TP53, p27kip1 and c-myc gene expression in EpCAM transfected cells. Overexpression of EpCAM upregulated c-myc gene expression. (D) Western Blot analysis of EpCAM overexpression and upregulation of c-myc protein levels. Tubulin alpha served as internal loading control. (E) Densitometric analysis of c-myc to tubulin protein ratio. MCF10A cell lines were generated by a lentiviral system to have a stable expression of a non-silencing control (ns/crtl) or an EpCAM specific (E#2) shRNA. MCF10A ns/crtl and MCF10A E#2 cells were adenovirally transfected to overexpress GFP or EpCAM/GFP. (F) In comparison to MCF10A ns/crtl cells MCF10A E#2 cells were significantly downregulating EpCAM transcript levels 24 and 48 h after adenoviral transfection. (G) Real time cell proliferation of MCF10A E#2 cells was significantly lower than those of MCF10A ns/crtl after adenoviral EpCAM overexpression. Stars indicate p values <0.05.