Levels and localization of tight junction protein Claudin-7 are regulated by EpCAM in MDCK cells.
(A) EpCAM-depleted MDCK lines Esh1, Esh2 and control shRNA line ctrl sh were SDS extracted one day after plating at subconfluent cell density. Protein levels of Claudin-7 and of GAPDH as a loading control were analyzed in the same immunoblot. The graphs show quantification of Claudin-7 protein normalized to GAPDH levels in the same sample. Error bars: S.E.M. of four samples for each cell line; p values derived from unpaired Student’s t test: ** p = 0.0042 for Esh1 to ctrl sh and ** p = 0.0057 for Esh2 to ctrl sh. (B) Claudin-7 protein levels in MDCK cells and cell lines MhE16, MhE33 overexpressing human EpCAM were analyzed as described in (A). Arbitrary units for protein intensities in Y-axis (AU) x103; error bars: S.E.M. of three samples for each cell line; p value derived from unpaired Student’s t test: * p = 0.042 for MhE16 to MDCK. (C) Confocal images were acquired of cells 24 hours after plating on IBIDI chambers and 0 hours after removal of the stencil. The figure shows maximum intensity projections and orthogonal projections of confocal stacks of MDCK, MhE16 and Esh2 cells stained for nuclei (blue), EpCAM (green), and Claudin-7 (red). In the MDCK and MhE16 lines, both EpCAM and Claudin-7 localize along basolateral membrane. In the Esh2 line, Claudin-7 does not distribute throughout the basolateral membrane, and EpCAM staining is very weak. In Esh2 line, greater contrast was applied post-processing for clarity. Nuclear signal in EpCAM channel in Esh2 is due to residual mCherry signal in the nucleus post methanol fixation. Scale bar is 10μm.