Rescue of Claudin-7 protein levels by expression of EpCAM-YFP or point mutants of EpCAM-YFP in MDCK cells depleted of EpCAM.
(A) Schematic of EpCAM-YFP (EY) and EpCAM-YFP mutant proteins (EIY and EEY). Extracellular domain (EC), transmembrane domain (TM), intracellular domain (IC) and YFP fusion are indicated. Approximate position of amino acids mutated in EIY and EEY are indicated with their one letter codes. (B) EpCAM-depleted Esh2-derived cell lines expressing EY, EIY or EEY were plated at confluent density onto an IBIDI 4 well dish for one day and YFP fluorescence was imaged before well chambers were removed for a migration assay. YFP fluorescence localizes to cell-cell contacts in all three cell lines. Scale bars: 50 μm. (C) Indicated MDCK cell lines were cultured, extracted and protein levels were analyzed as described for Figs 1A, 2A and 5. EpCAM-YFP fusion proteins EY, EIY and EEY appear as double bands; MDCK-endogenous EpCAM (ME) is a single band. EEY appears slightly larger and is poorly recognized by the EpCAM extracellular domain antibody (top EpCAM blot). Expression of Claudin-7 is reduced in the parental Esh2 line (Esh) compared to MDCK and control shRNA line ctrl sh (Claudin-7 blot; see also Fig 5). (D) Graphs show quantifications of indicated proteins normalized to GAPDH levels in the same sample. For EpCAM protein levels, residual MDCK-endogenous EpCAM (ME) and EpCAM-YFP protein double band levels (EY, EIY, EEY) were combined to show total EpCAM levels in the Esh2 lines expressing EY, EIY or EEY. Total EpCAM is ~ 20x or ~21x higher in Esh + EY and ~ 64x or ~68x higher in Esh + EIY compared to MDCK or ctrl sh cell lines. Please note that the EpCAM antibody was made against human EpCAM and may have different affinities for the endogenous canine EpCAM in MDCK and ctrl sh cell lines and the exogenous human EpCAM-YFP proteins, so these values are only approximations. Since EEY is poorly recognized by EpCAM antibody, expression levels of the three YFP fusion proteins are best compared in the YFP immunoblot (middle YFP blot). Error bars: S.E.M. of six samples for each cell line. Expression of EY, EIY or EEY in Esh 2 rescues Claudin-7 protein levels in these cell lines compared to Claudin-7 level in the Esh 2 line (Esh in Claudin-7 graph): p values are derived from unpaired Student’s t test; p values compared to MDCK: *** p = 0.0002 for Esh and *** p = 0.0005 for Esh+EEY; p values compared to ctrl sh: ** p = 0.0016 for Esh; * p = 0.0433 for Esh+EY, *** p = 0.0005 for Esh+EIY and *** p = 0.0001 for Esh+EEY. Esh 2 has significantly less Claudin-7 than MDCK and ctrl sh control lines; Esh+EY has Claudin-7 levels comparable to control lines; Esh+EIY and Esh+EEY have significantly more Claudin-7 than ctrl sh. (E) Claudin-7 immunoprecipitates (IP) of indicated MDCK cell lines were immunoblotted for EpCAM (top blot), YFP (middle blot) or Claudin-7 (bottom blot). ME in ctrl sh cells co-immunoprecipitates with Claudin-7 (top EpCAM blot) but ME is depleted in all Esh lines. EY in Esh+EY cells and EEY in Esh+EEY cells co-immunoprecipitate with Claudin-7 (top and middle blot for EY, middle blot for EEY). EEY is not recognized by EpCAM antibody in the EpCAM blot (E: top blot; see C, D) but by antibody to YFP (middle blot). EIY is mutated in the Claudin-7 interaction domain and does not co-immunoprecipitate with Claudin-7 from Esh+EIY cells (top and middle blot). Immunoglobulin heavy chain (Ig) from the Claudin-7 antibody used for the immunoprecipitation is recognized by secondary antibodies in the immunoblots. Immunoprecipitations were done twice and were repeated with an independent set of Esh lines showing the same results.