Gel densitometry

Total protein content was extracted using lysis buffer that contained (1%NP‐40, 20 mmol/L Tris, pH8.0, 137 mmol/L NaCl, 0.5 mmol/L EDTA, 0.1% sodium dodecyl sulphate [SDS], 10% glycerol, 10 mmol/L Na2P2O7, 10 mmol/L NaF, 1 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mmol/L sodium vanadate and 1 mmol/L PMSF). The protein concentration was estimated using bicinchoninic acid (BCA) protein assay (BCA1, Sigma‐Aldrich, St. Louis, MO, USA). The isolated proteins were separated by SDS‐polyacrylamide gel electrophoresis, and then transferred to polyvinylidene fluoride membrane. The membrane was probed with addition of primary anti‐rabbit antibodies against HEPH (1:200, ab108003), CDX2 (1:10 000, ab76541), GAPDH (1:2500, ab9485), α‐SMA (1:1000, ab32575), FAP (1:1000, ab53066), FSP‐1 (1:1000, ab124805), vimentin (1:1000, ab193555), CD63 (1:1000, ab134045), CD81 (1:1000, ab109201) and tumour susceptibility gene 101 (TSG101; 1:5000, ab125011) at the controlled temperature of 4°C overnight prior to re‐probing with secondary antibody. All of the preceding antibodies were acquired from Abcam (Cambridge, UK). The image was subjected to greyscale analysis using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Image density of the immunoblotting was subjected to determination with the application of Gel densitometry (Bio‐Rad, Hercules, CA, USA).

Cells were cultured at a density of 1.5 × 10⁵ cells/well on 8 mm coverslips in 12-well plates. After 48 hours, coverslips were fixed by ice-cold methanol, and incubated with primary E-cadherin (Abcam), Vimentin and β-catenin (Epitomics) antibodies prior to florescent-labeled secondary antibodies. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI) and coverslips were mounted with FluorSave reagent (CALBIOCHEM). Immunofluorescence images were taken by Olympus inverted fluorescence microscope and were outputted by PV10-ASW 1.7 viewer software. Immunoblotting was performed as follows: Proteins were extracted with lysis buffer and then quantified by the BCA method (KeyGen Biotech). Lysates were diluted in SDS sample buffer (KeyGen Biotech) prior to SDS-PAGE, and then transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences). Membranes were immunoblotted overnight at 4°C with anti-SOX17 (Millipore), anti-CyclinD1 (Abclonal), anti-C-myc, anti-DKK1 (Cell Signaling Technology), anti-E-cadherin (Abcam), anti-SOX2, anti-β-catenin, anti-Vimentin and anti-Slug and anti-N-cadherin antibodies (Epitomics), followed by the appropriate second antibodies. The bands were exposed using Pierce ECL Western Blotting Substrate (Thermo Scientific). Gel densitometry (Bio-Rad) was used to quantify immunoblot signals on exposed film.

Proteins were extracted with a lysis buffer and then quantified by a bicinchoninic acid protein assay. Equivalent amounts of cell lysates were separated using SDS‐PAGE and transferred to a polyvinylidene difluoride membrane (Roche Applied Sciences, Indianapolis, IA, USA). Membranes were immunoblotted overnight at 4°C with corresponding antibodies (Table S1). The bands were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Image density of the immunoblotting was determined by Gel densitometry (Bio‐Rad).