Vimentin

Total protein was extracted by lysing the cells with RIPA buffer and protease inhibitor. After denatured, proteins were run in the 10% SDS-PAGE gel and transferred to PVDF membranes. Membranes were blocked in 5% skim milk for 1 h at room temperature. Primary antibodies, vimentin (1:3000, abcam, Cambridge, MA, USA), E-cadherin (1:3000, abcam) and ZEB1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated overnight at 4°C. After washed in TBST, membranes were incubated with anti-mouse or anti-rabbit antibodies (1:3000) at room temperature for 1 h. Protein bands were visualized by the ECL system (Millipore).
For immunofluorescence assay, cells were seeded in 24-wells plate. The next day, attached cells were fixed by 4% paraformaldehyde for 30 min, and penetrated by 0.5% Triton X-100 for 15 min, then blocked by 3% BSA for 1 h. Primary antibodies in 1% BSA, vimentin (1:300, abcam), ZEB1 (1:300, Santa cruz), were incubated overnight at 4°C. Then anti-mouse or anti-rabbit IgG FITC (1:500) were incubated in at room temperature for 1 h and then stained with DAPI. Finally, coverslips were observed under a fluorescence microscope.

The samples on the nanofabricated coverslip were fixed with ice-cold 4% paraformaldehyde for 20 min, washed two times with phosphate-buffered saline (PBS), and permeablized with 0.1% Triton X-100 in PBS for 5 min. After washing with PBS, cultures were blocked with 10% goat serum for 1 h, and then incubated with primary antibodies against Vinculin (1:200, Sigma-Aldrich), E-cadherin (1:200, Cell Signaling, 3195), phospho-Myosin Light Chain (1:200, Cell Signaling, 3674), γ-tubulin (1:100, Abcam, ab11321), YAP (1:100, Cell Signaling, 4912 and Santa Cruz, sc-15407), active β-catenin (1:100, Millipore, 05-665), Vimentin (1:100, Abcam), Slug (1:100, Cell Signaling, 9585), Twist (1:100, Santa Cruz, sc-15393), Vimentin (1:100, Abcam, ab24525), and WT1 (1:100, Santa Cruz, sc-192) for 3 h at room temperature. After washing with secondary antibodies and Alexa Fluor 594 conjugated phalloidin (1:40, Molecular Probes) and Hoescht (Invitrogen), cultures were incubated for 1 h at room temperature. The slides were mounted with an anti-fade reagent (SlowFade gold, Invitrogen) and taken by an inverted microscope (Zeiss Axiovert 200 M) with an X40 oil immersion objective (Zeiss, 1.6 NA).

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).

The tissues were cryopreserved under the −80 °C and recovered at room temperature for 5 min. Tissues were then fixed in ice acetone at room temperature for 10 min, washed three times with PBS and blocked in 3% BSA at 37 °C for 1 h. The tissues were then incubated with E-cadherin (1:20) (BD Biosciences), N-cadherin (1:200) (Santa Cruz Biotechnology) and vimentin (1:5000) (Epitomics) antibodies overnight at 4 °C and washed three times with PBS immediately when transferred to room temperature. Then, the tissues were stained with Alexa Fluor 488- and 594-conjugated (Invitrogen) secondary antibodies at 37 °C for 1 h in a dark box and washed three times with PBS; the nuclei of the tissues were counterstained with prolong gold anti-fade reagent (Invitrogen). All images were captured with confocal laser scanning microscopy (Olympus FV1000).

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.

Antibodies: EZH2 (Cell Signaling Technology 3147), Actin (MAB1501), Total Histone H3 (Cell Signaling Technology 3638), H3K27me3 (Cell Signaling Technology 9733), H3K27me2 (Cell Signaling Technology 9728), MLC2 (Cell Signaling Technology 3672), pMLC2 (ser 19) (Cell Signaling Technology 3671), E-Cadherin (BD Biosciences 610182), and Vimentin (Epitomics 4211–1).

Tissue samples were cut into 2-3-mm pieces and homogenized in lysis buffer (1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 1 mM EGTA, and protease and phosphatase inhibitors) using a homogenizer on an ice tray, and the protein concentration was determined by BCA reagent. Protein samples were mixed with sample buffer, boiled for 5 min and separated by SDS–PAGE. Proteins on gel were then transferred onto PVDF membrane, blocked in blocking buffer containing 5% BSA, and then probed with primary antibodies against E-cadherin, Rab11, vimentin ( Epitomics, CA, USA) or GAPDH (Santa Cruz, CA, USA ), followed by incubation with appropriate HRP-conjugated secondary antibodies. Blots were developed using an enhanced chemiluminescence system.