EpCAM-depletion promotes cell spreading and epithelial sheet migration.
(A) SDS extracts from MDCK cells or MDCK cell lines expressing either control shRNA (ctrl sh) or anti-EpCAM shRNA 1 or 2 (Esh1; Esh2) were immunoblotted for EpCAM and GAPDH. Graph shows quantification of EpCAM protein levels normalized to GAPDH protein levels in the same samples. Error bars: S.E.M. of three samples for each cell line. Compared to parental MDCK cells, EpCAM was 5x reduced in Esh1 and 15x reduced in Esh2 expressing cells; compared to ctrl sh EpCAM was 4x reduced in Esh1 and 11x reduced in Esh2 expressing cells. (B) Phase images (top) and quantification of area/cell (graph) of individual colonies of indicated cell lines. Scale bars: 20 μm. Area per cell was significantly increased in EpCAM depleted cell lines Esh1 and Esh2 with ***p values < 0.0001 (two-tailed Mann-Whitney) compared to MDCK or ctrl sh lines. Error bars: S.E.M. of 90 MDCK, 88 ctrl sh, 104 Esh1 and 58 Esh2, small colonies of 5 to 200 cells (C) Phase images of indicated cell lines at 0 hrs (top row) and at 6 hrs migration (middle row). The bottom row shows an overlay of “6 hrs” images onto “0 hrs” images. Scale bars: 100 μm. Graph in (C) shows quantification of average change of area/image after 6 hrs migration for each cell line. Error bars: S.E.M. of nine (MDCK), four (ctrl sh, Esh1) and three (Esh2) experiments. Stars correspond to p values derived from an unpaired Student’s t-test. For Esh1 p = 0.034 compared to MDCK and p = 0.003 compared to ctrl sh; and for Esh2 p< 0.001 compared to MDCK or ctrl sh.