Fig 2: SILAC- and mass spectrometry-based identification of potential interactors of murine EpCAM(A) YFP (upper panels) and EpCAM-YFP (lower panels) were visualized by confocal laser scanning microscopy in stable mF9 cell transfectants expressing YFP (top panels) or EpCAM-YFP (lower panels) in the absence of any additional treatment. Nucleic DNA was visualized with Hoechst 33,342 (left panels). Shown are representative images. Scale bar represents 20 μm.(B) Schematic representation of SILAC screening performed with labeling with heavy (Lys-8/Arg-10) and light amino acids (Lys-0/Arg-0). YFP fusion proteins and interacting proteins were immunoprecipitated with a GFP-trap, pooled, and proteins analyzed upon liquid chromatography and mass spectrometry (LC-MS/MS).(C) Interactome of all known protein-protein interactions between 77 EpCAM partners identified in mF9 cells through SILAC-IP-MS (n = 3; one-tailed t test, permutation-based false discovery rate (FDR) < 0.05) visualized with the STRING database and R-package igraph. Hierarchical cluster analysis of the interactome was performed, and interaction clusters were formed based on cluster edge-betweenness. Nodes describe (1) the enrichment ratio (size, 3–61.49); (2) interaction clusters (color); (3) network edges represent known functional interactions in the STRING database.

Fig 3: Classification of potential interactors of EpCAM(A and B) Two potential interactors of EpCAM with high enrichment scores (Prohibitin 1 and 2 Phb1 and Phb2) and one potential interactor with lower enrichment score (Calnexin, Caln) were co-precipitated with EpCAM-YFP but not YFP in stable transfectants of mF9 teratocarcinoma cells (A) and in E14TG2α murine embryonic stem cells (B). Phb1, Phb2, and Caln levels were comparable in whole-cell lysates of EpCAM-YFP and YFP stable transfectants of mF9 and E14TG2α. Amounts of EpCAM-YFP and YFP following immunoprecipitation with GFP-Trap® agarose beads were controlled with GFP-specific antibodies and were comparable. Shown are each one representative immunoblot from three independent experiments.(C and D) KEGG Pathways and Gene Ontology (GO) terms “Biological process” (BP) to analyze potential interactors of EpCAM. KEGG pathways and GO terms are depicted with the protein counts in each set and false-discovery rate (FDR) ≤ 0.05.(E) Potential interactors of EpCAM (n = 59/77) were implemented on the left side of a chord diagram with their respective enrichment ratio and the GO terms in which they feed into on the right side of the chord diagram.

Fig 4: EpCAM localizes to intracellular vesicles(A) Spherical network of SILAC potential interactors of EpCAM found in the GO BP term “vesicle-mediated transport”. Degree of interactions encodes node size. Color shows the enrichment ratio from low (blue) to high (red). Arrow indicates direction of interaction.(B) mF9 cells stably transfected with EpCAM-YFP, mEpICD-YFP, or YFP were treated with DMSO or the V-ATPase inhibitor bafilomycin A1 (10 nM). Bright field and YFP fluorescence were visualized by confocal laser scanning microscopy. Shown are representative pictures. Scale bars represents 20 μm.(C) mF9 cells stably transfected with EpCAM-YFP, EpICD-YFP, or YFP were treated with DMSO or bafilomycin A1. Mean fluorescence intensity ratios were assessed by flow cytometry. Shown are the results from 3 to 4 independent experiments. Mean values are indicated by a line. p value: ∗∗ ≤0.01, n.s.: not significant.

Fig 5: Endocytosis of EpCAM in mF9 teratoma cells(A) mF9 cells stably transfected with EpCAM-YFP and transiently transfected with m-Cherry-tagged Rab5, Rab7, and Rab11 were visualized by laser scanning confocal microscopy. Lysosomes were detected with Lysotracker (Red DND-99 Ex577/Em590 nm). Cells were treated with either DMSO or 10 nM bafilomycin A1 (10 nM) as indicated. Scale bars represent: Rab5 5 μm; Rab7 and Rab11 20 μm (DMSO) and 5 μm (Bafilomycin); Lysotracker: 20 μm. Brightness and contrast of both channels were adjusted linearly.(B) Co-localizations of EpCAM-YFP with the indicated mCherry-tagged Rab proteins and lysosomes were assessed as indicated in STAR Methods. Shown are Manders' coefficients representing the fraction of mCherry-tagged Rab protein or lysotracker overlapping with EpCAM-YfP as dot plots with mean and SD from each n = 7 and n = 2–3 independent imaging areas for bafilomycin A1-treated cells and DMSO-treated cells, respectively.(C) Whole-cell lysates from mF9 cells stably transfected with EpCAM-YFP or YFP were immunoprecipitated with GFP-Trap® agarose beads. Immunoprecipitated proteins were separated by SDS-PAGE and detected with anti-YFP, anti-Rab5, anti-Rab7, and anti-Rab11 antibodies in combination with HRP-conjugated secondary antibodies. Additionally, whole-cell lysates were separated by SDS-PAGE, and Rab5, Rab7, and Rab11 proteins were detected with specific antibodies in combination with HRP-conjugated secondary antibodies. Shown are representative results from three independent experiments.(D) Schematic representation of the endocytosis and membrane recycling assay.(E) Kinetics of EpCAM endocytosis (left panel) and membrane recycling (right panel). Shown are mean percentages of EpCAM endocytosis and membrane recycling with SD over a time of 30 min from three independent experiments. One-way ANOVA with Dunnet's multiple tests. ∗∗∗∗ p value < 0.0001.