Hecd 1

Tumor tissues in the PDX assays and MiniPDX assays were fixed in buffered 10% formalin and routinely stained with hematoxylin and eosin (H&E) and examined by a certified pathologist.
For immunofluorescence studies, cellularized tumor cells (2 × 10⁴ cells, 200 L) were cytospun onto a slide, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.3% Triton X-100 in PBS for 30 min, and then blocked with 5% normal goat serum for 1 h at room temperature. The cells were then divided into three fractions and incubated with primary mouse monoclonal antibodies at 4 °C overnight against the following proteins: pan-cytokeratin, indicating carcinoma components [27 (link), 28 (link)] (1:200, AE1/AE3, sc-81714, Santa Cruz Biotechnology, Santa Cruz, CA, US), E-cadherin, generally found in gastric adenocarcinomas [29 (link)] (1:50, HECD-1, ab1416, Abcam, Cambridge, UK), and MG7, a marker of gastric cancer [30 (link)] (1:300, NOTA-MG7) [30 (link)]. Subsequently, the cells were probed with secondary antibody donkey anti-mouse IgG H&L (Alexa Fluor^® 488) (1:200, ab150105, Abcam). Finally, the cells were mounted with DAPI-containing mounting medium (S36973, Thermo Fisher, MA, US). Images were captured with a fluorescence microscope (Leica, Germany) with Leica Application Suite V4 software and edited with Photoshop (Adobe, US).

Akt2 (5B5), p-AKT (S473; 736E11), p44/42 MAPK (Erk1/2; 137F5), p-p44/42 MAPK (T202/Y204, D13.14.4E), p-Smad2 (S465/467; 138D4), Smad4, SNAI2, ZEB1 (all Cell Signaling Technology); AGR2 (K-31, in-house); AGR2 (1C3, Abnova); vimentin (V9, Dako); E-cadherin (NCH38, Dako; HECD1, Abcam; Cell Signaling); N-cadherin (3B9, Invitrogen); β-actin (C4, Santa Cruz Biotech); Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 532 goat anti-rabbit IgG (both Abcam); α-tubulin (AA13, Sigma); HRP-conjugated swine anti-rabbit and HRP-conjugated rabbit anti-mouse (both Dako).

Co-localization between ECAD and EPN3 (Fig. 5c) was performed using an anti-ECAD Ab (HECD-1) from Abcam, recognizing the extracellular domain of ECAD^{66 (link)}. Briefly, MCF10A-Ctr or -EPN3 cells were plated on coverslips at ~30% confluence. Cells were starved overnight in culture medium containing 5% horse serum in absence of EGF. After starvation, coverslips were washed with PBS and incubated with the anti-ECAD Ab (2 μg ml⁻¹) for 1 h at 4 °C. The time zero sample was immediately fixed with 4% PFA. The other samples were incubated at 37 °C for 90 min with culture medium containing 5% horse serum and TGFβ (5 ng ml⁻¹). After incubation cells were fixed with 4% PFA, followed by IF (as described in the Paragraph: “ECAD staining at the cell-to-cell junction”). Cells were stained with anti-EPN3 rabbit polyclonal Ab (50 ng ml⁻¹). EPN3 and ECAD signals were then revealed with secondary Ab Cy3-conjugated (red, donkey anti-rabbit IgG 715-165-152, Jackson ImmunoResearch, dilution 1:400) or Alexa-488-conjugated (green, donkey anti-mouse IgG 715-165-150, Jackson ImmunoResearch, dilution 1:400), respectively. Images were obtained using a Leica TCS SP2 or TCS SP2 AOBS confocal microscope equipped with a 63× oil objective and processed using ImageJ software (v1.52, NIH).