Figure 4

EpCAM inhibits TGF-β1 induced terminal growth arrest and differentiation in HMECs. Adenovirally transfected HMECs were stimulated with 1 ng/mL TGF-β1 to undergo terminal growth arrest and differentiation in vitro. (A) In comparison to control cells TGF-β1 treated GFP expressing cells got growth arrested, flat and enlarged. Populations of EpCAM transfected cells were protected from TGF-β1 and acquired a small cell body (white arrows). (B) In comparison to proliferating control cells TGF-β1 treated cells stained positive for senescence-associated beta galactosidase (SA-β-Gal, blue color), a marker for terminally arrested cells. EpCAM transfected cells were predominantly negative and acquired a more spindle shaped morphology (black arrows). (C) Long term cultures of transfected HMECs in the presence of TGF-β1. EpCAM transfected cells showed a higher proliferative capacity within the observed time window of 6 days than GFP controls. Bars indicate 25 μm. (D) Cell numbers were analyzed 1, 3 and 6 days after TGF-β stimulation by counting in a Buerker-Tuerk chamber. EpCAM transfected cells displayed significantly higher proliferative activities, i.e. higher cell counts after 6 days of growth. (E) Western Blot analysis of differentiation markers for epithelial mesenchymal transition (vimentin, E-cadherin). (F) EpCAM transfected HMECs show a downregulation of E-cadherin (E-cad) but no significant upregulation of vimentin (Vim) protein. Stars indicate p values <0.05.