Vimentin

Western blotting was performed as previously described [32 (link)]. Immunoblot was carried using the following primary antibodies: ELK1 and STRN4 (Abcam); Vimentin (R&D); CyclinD1 (Santa Cruz); E-Cadherin and N-Cadherin (BD); α-E-catenin, ERK1/2 and p-ERK (Cell Signaling); β-actin (Thermo Fisher).

Analysis of protein expression was performed by immunoblotting as described previously [7 ,8 (link)]. Western blots and were probed with the following antibodies: NRF1 (Rockland Immunochemicals Inc., Pottstown, PA, USA), Nanog (Cell Signaling Technology, Inc., Danvers, MA, USA), Vimentin (cat# AF2105, R&D System, Minneapolis, MN, USA), E-Cadherin (cat# ab15148, Abcam, Cambridge, MA, USA), N-Cadherin (cat# sc-8424, D-4, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), CXCR4 (cat# sc-9046, H-118, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ALDH1 (cat # sc-22589, L-15, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and β-actin (13E5, rabbit mAb #4970, Cell Signaling Technology, Inc.). Electrochemiluminescence (ECL) intensity of detected target proteins was imaged and quantified with a Bio-Rad Versa Doc instrument. All immunoblots were completed a minimum of three times for each experiment.

Staining was performed on established cell lines growing in chambers of the Nunc Lab-Tek Chamber Slide system (Sigma-Aldrich Chemie), or on 6 μm frozen or paraffin-embedded tissue sections as previously described.^{50 (link)} The antibodies were mouse monoclonal against cytokeratin (Cyt)-19 (Abcam), rabbit monoclonal against E-cadherin (Cell Signaling Technology), Ki-67 (Abcam), Notch1, P-cJun (Cell Signaling), goat polyclonal against vimentin (R&D Systems, Wiesbaden-Nordenstadt, Germany), SOX2 (Santa Cruz), c-Met (Biozol, Eching, Germany) and TGFβ1 (Acris, Herford, Germany).

Recombinant osteopontin was purchased from R&D Systems (Minneapolis, MN, USA, #1433-OP-050/CF), ACROBiosystems (Newark, DE, #OPN-H5227), and Abcam (Cambridge, UK, #ab92964, #ab281819). Fibronectin, tenascin-C, bone sialoprotein, and vimentin were purchased from R&D Systems (Minneapolis, MN, USA). a-enolase and type II collagen were purchased from Abcam (Cambridge, UK). Fibrinogen was purchased from the Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Antibodies against total focal adhesion kinase (FAK) and phosphorylated FAK were purchased from Cell Signaling Technology (Danvers, MA, USA, #3285 and #8556). Recombinant human tumor necrosis factor (TNF) was purchased from Peprotech (Cranbury, NJ, USA, #300-01A). The secondary antibody used for immunoblot analysis was peroxidase-conjugated anti-rabbit IgG (Agilent, Santa Clara, CA, USA; #P0399).

Western blotting was performed as described previously49 (link). Cell lysates (10 μg) were subjected to SDS–polyacrylamide gel and immunoblot analysis with antibodies against the following proteins: HMGA2, Sox2 and Vimentin (R&D); SALL4 and Oct4 (Protein Tech.); Nanog, Bmi-1 and β-actin (Millipore); Twsit1, LOX and Fibronectin (Abcam); p-FAK (Invitrogen); E-Cadherin, N-Cadherin and β-catenin (BD); α-E-catenin (Cell Signaling); a-SMA (Sigma Aldrich).

Fetal bovine serum (FBS), antibiotics (penicillin-streptomycin), and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco-BRL (Grand Island, NY, USA); 3-(4, 5-dimethylthiazol-2-yl)-2; dimethyl sulfoxide (DMSO) and in situ Cell Death Detection Kit (TMR red) from Sigma-Aldrich (St. Louis, MO, USA); 5-diphenyltetrazolium bromide (MTT) from Molecular Probes (Eugene, OR, USA). The following antibodies were used: GAPDH, Bcl-2, survivin, caspase-3, caspase-8, cleaved caspase-3, PCNA and p53 (Cell Signalling, Danvers, MA, USA), PARP, and Bcl-xL (Santa Cruz Biotechnology, Dallas, TX, USA), matrix metalloproteinase (MMP)-2 (Bioss Antibodies Inc., Woburn, Massachusetts, USA), MMP-9, Ki-67, and alexa 488/594 (Abcam, Cambridge, UK), and p21, cyclin D1, CDK4, Bak, tBID, E-cadherin, vimentin, and snail (R&D Systems, Minneapolis, MN, USA). Annexin V-FITC Apoptosis Detection Kit, propidium iodide (PI), Hoechst 33342 staining kit, Matrigel, and various caspase inhibitors were purchased from BD Biosciences (San Jose, CA, USA).

5 µm joints sections were deparaffinized and antigens retrieved by heat (10 mM sodium citrate, 0.05% Tween20, pH6; 60 min 65 °C) or proteinase K (5 min 37 °C). Endogenous peroxidases were blocked with 3% H₂O₂ in methanol, unspecific bindings with 5% BSA. Primary antibodies: leptin (ab3583, abcam, Cambridge, UK), visfatin/PBEF (H-300, sc-67020, Santa Cruz, Dallas, USA), adiponectin/Acrp30 (AF1119, R&D Systems, Minneapolis, USA), F4/80 (abdserotec, Hercules, USA), CD45 (30-F11, Novusbio, Littleton, USA), collagen type X (Abbiotec, San Diego, USA), vimentin (R&D). Secondary antibody system: Histofine^® (Nichirei Biosciences, Tokyo, Japan). Isotype and negative controls were performed.