N cadherin

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).

SW480 and HCT116 cells were collected and lysed in RIPA buffer. Aliquots (25 μg) of total or nucleus protein were boiled with RIPA buffer and loaded onto 8% or 10% polyacrylamide gels and transferred to PVDF membrane (Immobilon-P, Millipore). Membranes were blocked for nonspecific antibody binding in 5% nonfat milk and incubated with the corresponding primary and secondary antibodies. GAPDH antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). hTERT, ZEB1, E-cadherin, N-cadherin and vimentin antibodies were obtained from Epitomics (Abcam, Cambridge, UK).

Transfected cells were lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl PH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1μg/ml leupeptin, 1 mM deoxycholic acid and 1 mM EDTA) containing a cocktail of protease inhibitors and phosphatase inhibitors (Calbiochem, Darmstadt, Germany). Equal amounts of protein sample (30-50 μg) were separated by 12% SDS-PAGE and transferred to PVDF membrane (Millipore, Bedford, MA, USA) using the Bio-Rad semidry transfer system. The following antibodies were used for Western blotting: IGF-1R, N-cadherin, CyclinD1 (Epitomics, USA); Bcl-xl (CST, USA), GAPDH and β-actin (Biostar, China).

Analytical 12% SDS-PAGE was performed, and 30 μg of protein were analyzed for each condition, unless otherwise stated. For immunoblotting, proteins in the SDS gels were transferred to a polyvinylidene difluoride membrane using an electroblot apparatus. Antibodies against human fibronectin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), FAK (Cell Signaling Technology, Danvers, MA), p-FAK (Epitomics, Burlingame, CA, USA), N-cadherin (Epitomics, Burlingame, CA, USA), E-cadherin (Epitomics, Burlingame, CA, USA), Snail1 (bs-1371R; Bioss, Boston, MA, USA), COX-2 (Lab Vision Corp., Fremont, CA), c-Jun (Santa Cruz Biotechnology), AKT and p-AKT (both from Cell Signaling Technology, Danvers, MA), p-Rac1/cdc42 (Cell Signaling Technology), and α-tubulin and β-actin (both from Sigma-Aldrich) were used as the primary antibodies. Mouse or rabbit IgG antibodies coupled to horseradish peroxidase were used as secondary antibodies. An enhanced chemiluminescence kit (Supersignal West Pico Chemiluminescence kit; Pierce, Rockford, IL) was used for detection. The FAK inhibitor Y15 was purchased from Sigma-Aldrich (St. Louis, MO).

When 80% confluence in 25 cm² flasks (Nunc, Roskilde, Denmark) was reached, the cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) on ice and then centrifuged at 12000 rpm for 20 min. Supernatants were collected and protein was determined using bicinchoninic acid (BCA) kit (Boster, Wuhan, China). The extracts (20 μg per lane) were fractionated by 10% SDS-PAGE and then transferred onto PVDF membranes (0.45 μm; Millipore, Bedford, MA). After blocking with TBST buffer (20 mM Tris-buffered saline and 0.1% Tween-20) containing 5% nonfat milk for 1 h at 25°C, the membranes were incubated with primary antibodies against E-cadherin, N-cadherin (1 : 5000, Epitomics, USA), cytokeratin (1 : 400, Boster, China), EP300 (1 : 1000, Abnova, USA), and β-actin (1 : 5000, CMCTAG, USA) overnight at 4°C. Membranes were washed three times for 5 min with TBST before incubation with horseradish peroxidase-conjugated secondary antibody (1 : 3000, CST, USA) for 60 min at 25°C. The membranes were exposed by chemiluminescence (Millipore, Billerica, MA), and images were acquired by ChemiDoc XRS (Bio-Rad, Hercules, CA). Semiquantification of scanned films was performed using Quantity One-4.6.2 (Bio-Rad).

Cells were lysed in RIPA lysis buffer (Beyotime, Nanjing, China) with the protease inhibitor phenylmethanesulfonyl fluoride (Beyotime). Protein concentrations were determined using a BCA Protein Assay kit (Beyotime). Equal amounts of protein were loaded into each lane of an SDS-PAGE gel. Proteins were then separated with electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Each membrane was blocked with 5% skimmed milk for 2 h and then incubated with antibodies against SHARP1 (1:500), E-cadherin (1:1000), N-cadherin (1:1000), vimentin (1:1000), Snail (1:1000), NOTCH1 (1:2500; Epitomics, Burlingame, CA), jagged 1 (1:10000), HES1 (1:2000; Epitomics), or β-actin (1:5000; Epitomics) at 4°C overnight. Peroxidase-linked secondary antibodies against rabbit (1:5000; Epitomics) were used to detect bound primary antibodies. Probed proteins were detected by enhanced chemiluminescent reagents. The data have been normalized to β-actin expression by densitometry and statistical data from at least 3 experiments is graphed to provide additional validation of results.

Whole-cell lysates were separated in 12% SDS-PAGE gels and blotted on nitrocellulose membranes, and probed with antibodies against β-Actin (Santa Cruz Biotechnology, Inc.), RhoGDI1, Fibronectin, Snail (Proteintech), N-cadherin (Epitomics), E-cadherin, and β-catenin (Cell Signaling Technology). After incubation with primary antibodies, the membranes were washed with TBS/0.05% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Signals were detected using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA).