Epcam

Western blot analysis was performed using specific primary antibodies against CD133 (Proteintech, USA), CD44 (CST, USA), Lgr5 (Abcam, USA), EpCAM (Abcam, USA), ALDH1 (Abcam, USA), β-catenin (Proteintech, USA), Nanog (CST, USA), E-cadherin (CST, USA), N-cadherin (Epitomics, USA), vimentin (CST, USA), Snail (Abcam, USA), Twist (Abcam, USA), Slug (Abcam, USA), ZEB1 (Abcam, USA), fibronectin (Abcam, USA), Sox2 (CST, USA), Oct4 (CST, USA), PRDX2 (Abcam, USA), Cyclin D1 (Abcam, USA), c-Myc (Abcam, USA), MMP-2 (Abcam, USA), MMP-9 (Abcam, USA), VEGF (Abcam, USA) and GAPDH (Goodhere, China).

Cells were lysed in lysis buffer (1% NP-40, 50 mM Tris, 0.1% SDS, 1 mM PMSF, 10 mM EDTA, 150 mM NaCl, and 0.5% sodium deoxycholate) as instructed (Beyotime, China). Supernatants of lysates were collected after centrifugation. A BCA protein detection kit (Beyotime, China) was used to determine the protein concentration. SDS/PAGE was used for the separation of the indicated amounts and then transferred on to PVDF membranes (Millipore, U.S.A.). Using nonfat milk, the membranes were blocked for 1 h. Next, primary antibodies were incubated overnight at 4°C. Then, the membranes were washed using TBST for 15 min and incubated with secondary antibodies (1:5000) at 37°C for 1 h. After washing with TBST for 15 min, the detection was performed with Fusion FX (Vilber, France) using an enhanced chemiluminescence kit (Millipore, U.S.A.). Specific bands were quantified using Fusion software. Each experiment was performed in triplicate. The following antibodies were used: CD133 (Proteintech, U.S.A.), CD44 (CST, U.S.A.), Lgr5 (Abcam, U.S.A.), EpCAM (Abcam, U.S.A.), ALDH1 (Abcam, U.S.A.), β-catenin (Proteintech, U.S.A.), CD166 (CST, U.S.A.), E-cadherin (CST, U.S.A.), N-cadherin (Epitomics, U.S.A.), vimentin (CST, U.S.A.), Snail (Abcam, U.S.A.), Twist (Abcam, U.S.A.), Slug (Abcam, U.S.A.), ZEB1 (Abcam, U.S.A.), fibronectin (Abcam, U.S.A.), and GAPDH (Goodhere, China).

The following antibodies were used: N-cadherin (clone GC4, Sigma-Aldrich C3865, 1:100 dilution), FSP1 (Abcam ab58597, 1:100 dilution), PDGFRα (R&D Systems AF1062, 1:50 dilution), Collagen I (Rockland 600–401–103–0.1, 1:150 dilution), Collagen III (Abcam ab7778, 1:150 dilution), Fibronectin (Abcam ab23750, 1:200 dilution), α-SMA (Abcam ab5694, 1:100 dilution), CD31 (Novus Biologicals NB100-2284, 1:500 dilution), EpCAM (Abcam ab92382, 1:500 dilution), Lyve-1 (Abcam ab14917, 1:500 dilution), Myf-5 (clone C-20, Santa Cruz sc-302, 1:500 dilution), CD45 (clone IBL-3/16, Abcam ab23910, 1:500 dilution), FABP4 (clone EPR3579, Abcam ab92501, 1:500 dilution), F4/80 (Abcam ab90247, 1:500 dilution), Cytokeratin 14 (clone EPR17350, Abcam ab181595, 1:200 dilution), Integrin α_v (clone EPR16800, Abcam ab179475, 1:500 dilution), α1-catenin (clone EP1793Y, Abcam ab51032, 1:500 dilution), decorin (Abcam ab175404, 1:500 dilution), DLK-1 (Abcam ab21682, 1:200 dilution) and Ki67 (clone SP6, Abcam ab16667, 1:500 dilution). Fluorophore-conjugated secondary antibodies were purchased from Thermo Fisher Scientific (1:500 dilution). Exherin (ADH-1) was from TargetMol (T2637).

Western blotting was performed as previously reported [38 (link), 39 (link)]. Briefly, total protein extracts were obtained by lysing the cells in RIPA buffer (150 mM Sodium Chloride, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, and 50 mM TrisHCl pH 8.0) and proteins were quantified with the BCA method (Pierce, ThermoFisher Scientific); 20–50 μg of protein extracts were loaded on appropriate 7.5%, 10% or 12% polyacrylamide gels and SDS-PAGE was performed. Proteins were then transferred on Nitrocellulose membranes, which were probed over-night at 4 °C with specific antibodies diluted in 1% non-fat skim milk-PBS-T solution: ETV7/TEL2 (E-1, sc-374478), β-Tubulin (3F3-G2, sc-53140), STAT1 (D1K9Y, Cell Signaling Technology, Euroclone), p-STAT1 (Tyr701) (D4A7, Cell Signaling Technology), BCL-2 (100, sc-509), Survivin (D-8, sc-17779), EpCAM (ab71916, Abcam, Cambridge, UK), and HSP70 (C92F3A-5, sc-66048). Antibodies were obtained from Santa Cruz Biotechnologies (Heidelberg, Germany) when not explicitly indicated. Detection was performed with ECL Select reagent (GE Healthcare) using the UVITec Alliance LD2 (UVITec Cambridge, UK) imaging system.

Paraformaldehyde 4% (SIGMA-P6148) was used to fix the tissue samples. Following this, the fixed tissue samples were embedded in OCT and were cut with a Leica RM2255 microtome (Leica Biosystems; Wetzlar, Germany) into 10 um sections. Confocal scanning laser microscopy was used to analyse the antibody-stained sections with the use of a Leica SPE laser-scanning microscope (Leica Biosystems). Finally, the images were quantified with ImageJ.
Colorectal cancer tissue microarrays (TMAs) were generated with the obtained tissue samples. These arrays were stained for CD45, CD14 and EpCAM (all from Abcam). A mounting medium containing DAPI and α-fading (Vectashield; Vector Laboratories; Newark, CA, USA) was added to the sections.

ZER (chemical name: (2E, 6E.10E)-2,6,9,9-tetramethylcycloundeca-2,9,10-terien1-one), dimethyl sulfoxide (DMSO), and trypan blue were purchased from Sigma (St. Louis, MO, USA). ZER was dissolved with 1 mL DMSO to make a 229.35 mM stock solution and kept at –20 Cо. The working concentration of ZER was diluted in Dulbecco, s modified Eagle, s medium (DMEM) containing 4% FBS. The 5-FU was purchased from Sigma-Aldrich, and 50 mg of 5-FU was dissolved with 1 mL dH₂O to make a 384 mM stock solution and kept at room temperature. The future dilution was added in a DMEM medium containing 2% FBS to treated cells. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was prepared from Sigma Aldrich. Primary monoclonal antibodies against α-SMA, EPCAM, vimentin, survivin, and GAPDH were purchased from Abcam (Cambridge, AM, USA), and the secondary horseradish peroxidase (HRP)-conjugated, IgG-TR (Texas RED), and IgG FITC were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruze, CA). A high pure RNA isolation kit was obtained from Roche Applied Science (Germany), and SYBR Green QPCR Master Mix was purchased from Thermo Fisher Scientific (USA). The first-strand cDNA synthesis kit was obtained from Thermo Scientific (USA).

Monolayers cultured with and without latex microparticles were stained for Glycoprotein-2 (GP2) (MBL), E-cadherin (CDH1) (Dako), and Epithelial cell adhesion molecule (EPCAM) (Abcam). Epifluorescence microscopy was used to characterize differences between RANKL treated and non-treated groups. Confocal microscopy was used to characterize morphologic characteristics, as well as confirm z-stack co-localization of Texas Red latex microparticles within cells.