Figure 2

Lentiviral knockdown of EpCAM gene expression by shRNA in EpCAM ^high human breast cancer cell lines. (A) Lentiviruses were generated that express not only the green fluorescent protein (GFP) and the puromycin resistance gene (Puro), but also shRNAs targeting EpCAM (E#2) or a non-silencing control sequence (n/s ctrl). Transfected cells expressed turbo GFP and were selected with puromycin for five days. (B) The efficacy of EpCAM knockdown (E#2) in comparison to that of non-silencing controls (n/s ctrl) was proven by real-time PCR. Gene expression was reduced below 20% of the control value in all three cell lines analyzed. All cell types were analyzed in triplicate. (C) Knockdown of EpCAM was proven on the protein level by Western Blot analysis. Tubulin alpha served as internal loading control. In comparison to wild-type cells (wt) and n/s ctrl, EpCAM expression decreased in cells expressing E#2. Densitometric analysis of two independent experiments (means) as compared to wild type expression (100%). (D) Proliferation of transfected cells was monitored in real-time by the xCelligence system over a period of 72 h. E#2-expressing cells displayed significantly less proliferation and a smaller cell number when cultivated under serum-reduced conditions (n=6). (E) 3D growth of transfected cells was studied in collagen plugs by incorporating BrdU and subsequently measuring BrdU in a specific ELISA. E#2-expressing cells showed a significantly lower rate of DNA synthesis (n=6). Stars indicate p values < 0.05.