DLD1 colon carcinoma, MDCKII, and A375 melanoma cells were acquired from the American Tissue Culture Collection (Manassas, VA). Cells were grown in culture with RPMI 1640 medium for DLD1 and A375 cells or DMEM (Invitrogen, Carlsbad, CA) for MDCKII cells +10% fetal bovine serum +1% penicillin/streptomycin. Fetal bovine serum was removed for all experimental conditions. Recombinant TGF-β1, TGF-β2, or TGF-β3 (R&D Systems, Minneapolis, MN) was added to the culture medium at a concentration of 10 ng/ml for all relative experiments. Snail (provided by Dr. Angela Nieto, Cajal Institute, Madrid, Spain), Slug (provided by Dr. Tom Jessell, Columbia University, New York, NY), and pcDNA3-E-cadherin (provided by Dr. Barry Gumbiner, University of Virginia, Charlottesville, VA) expression plasmids (500 ng) were transfected into cells using Lipofectamine and Plus reagents (Invitrogen) according to the manufacturer's guidelines. Dominant-negative (DN) Smad4 (provided by Dr. Diane Simeone, University of Michigan, Ann Arbor, MI) and DN LEF-1 (provided by Dr. Marian Waterman, University of California, Irvine, CA) adenoviral constructs, which produce proteins that lack the ability to bind DNA, were added at dilutions of 1:100 as described previously (Nawshad and Hay, 2003
). Phosphoinositide-3-kinase (PI3K) inhibitor LY294002 (at a dilution of 1:50) and mitogen-activated protein kinase kinase (MEK)1/2 inhibitor U0126 (at a dilution of 1:20) (Cell Signaling Technology, Danvers, MA) were added for 1 h before treatment with TGF-β1, TGF-β2, or TGF-β3. TGF-β1/-β2/-β3 neutralizing antibody and TGF-β3 neutralizing antibody (R&D Systems), which does not cross-react with TGF-β1 or TGF-β2, were used according to the manufacturer's guidelines. Antisense oligodeoxynucleotides (Integrated DNA Technologies, Coralville, IA) were used at a concentration of 4.0 μg/ml and were synthesized using the following sequences: TCF-1, 5′-GAGTAGACGGTCTCTTTGTA-3′; LEF-1, 5′-CCTCCTCCGGAGAGTTGGGG-3′; TCF-3, 5′-CCCCCGGCGGCGAGCTGGGG-3′; TCF-4, 5′-CCACCGCCGTTCAGCTGCGG-3′; β-catenin, 5′-GTGGTCCACAGAACTTCTC-3′; and negative control, 5′-TTCCTCTCTTTTCTCTCCCT-3′.
RNA Interference (RNAi)
Small interfering RNA (siRNA) gene expression knockdown studies were performed using the TriFECTa RNAi kit (Integrated DNA Technologies) and corresponding protocol. Each 27mer RNAi duplex was transfected into cells using X-tremeGene siRNA transfection reagent (Roche Diagnostics, Indianapolis, IN) following the manufacturer's guidelines. siRNA was synthesized (Integrated DNA Technologies) using the following sequences: Snail, 5′-CCACAGAAAUGGCCAUGGGAAGGCCUC-3′; Slug, 5′-UCCGAAUAUGCAUCUUCAGGGCGCCCA-3′; LEF-1, 5′-CCGGGAUUUGCGCGCGGAGAACGCCGG-3′; and negative control, 5′-UCACAAGGGAGAGAAAGAGAGGAAGGA-3′.
Immunocytochemistry, Immunoprecipitation, and Immunoblotting
Immunofluorescence, immunoprecipitation, and Western blotting were performed using the following antibodies at concentrations (and using protocols) recommended by the respective manufacturers: TGF-β1, TGF-β2, TGF-β3 (R&D Systems), Smad4, TCF-1, LEF-1, TCF-3, TCF-4, Snail (Santa Cruz Biotechnology, Santa Cruz, CA), Slug (provided by Dr. Tom Jessell, Columbia University), β-catenin, vimentin, APC (Sigma-Aldrich, St. Louis, MO), P-Smad2/3, P-GSK-3β (Cell Signaling Technology), GSK-3β, fibronectin (BD Biosciences Transduction Laboratories, Lexington, KY), α-tubulin (Calbiochem, San Diego, CA). Horseradish peroxidase-conjugated secondary antibodies (Millipore Bioscience Research Reagents, Temecula, CA) were used at a dilution of 1:5000. Fluorescein- and rhodamine-conjugated secondary antibodies (Pierce Chemical, Rockford, IL) were used at a dilution of 1:250. Images were acquired using a Nikon 80i fluorescence microscope. Adjustments of image size, brightness, and contrast were made using Adobe Photoshop CS (Adobe Systems, Mountain View, CA).
Real-Time Quantitative Polymerase Chain Reaction (PCR)
RNA extractions were performed using the RNeasy Mini kit (QIAGEN, Valencia, CA) and protocol. RNA samples were submitted to a core facility (Biopolymers Facility, Department of Genetics, Harvard Medical School, Boston, MA) where real-time PCR experiments were conducted using the SYBER Green PCR system (Applied Biosystems, Foster City, CA) on an ABI 7500 cycler, with 40 cycles per sample. Cycling temperatures were as follows: denaturing, 95°C; annealing, 60°C; and extension, 70°C. The following primers were used: Snail, forward 5′-ACCACTATGCCGCGCTCTT-3′ and reverse, 5′-GGTCGTAGGGCTGCTGGAA-3′; Slug, forward 5′-TGTTGCAGTGAGGGCAAGAA-3′ and reverse 5′-GACCCTGGTTGCTTCAAGGA-3′; TGF-β3, forward 5′-AAGTGGGTCCATGAACCTAA-3′ and reverse 5′-GCTACATTTACAAGACTTCAC-3′; LEF-1, forward 5′-CCGAAGAGGAAGGCGATTTAGC-3′ and reverse 5′-GGTCCCTTGTTGTAGAGGCC-3′; Vimentin, forward 5′-TCTACGAGGAGGAGATGCGG-3′ and reverse 5′-GGTCAAGACGTGCCAGAGAC-3′; fibronectin, forward 5′-CCCACCGTCTCAACATGCTTAG-3′; reverse 5′-CTCGGCTTCCTCCATAACAAGTAC-3′; α-smooth muscle actin (SMA), forward 5′-CAATGGCTCTGGGCTCTGTAAG-3′ and reverse 5′-TGTTCTATCGGGTACTTCAGGGTC-3′; cyclin D1, forward 5′-ATGCCAACCTCCTCAACGAC-3′ and reverse, 5′-GGCTCTTTTTCACGGGCTCC-3′; α-actinin 1, forward 5′-GAAGAAATCCAGACCCTAGCACG-3′ and reverse 5′-GAGATGACCTCCAGCAGCAG-3′; E-cadherin, forward 5′-GTCAGTTCAGACTCCAGCCC-3′ and reverse 5′-AAATTCACTCTGCCCAGGACG-3′; Keratin 7, forward 5′-TCACCATTAACCAGAGCCTGC-3′ and reverse 5′-GGGCCTCAAAGATGTCTGGG-3′; and glyceraldehyde-3-phosphate dehydrogenase, forward 5′-ACCACAGTCCATGCCATCAC-3′ and reverse: 5′-TCCACCCTGTTGCTGTA-3′.
Chromatin Immunoprecipitation (ChIP)
The ChIP assay was performed using the ChIP-IT kit (Active Motif, Carlsbad, CA) and protocol. PCR analysis was performed on DNA isolated by ChIP using a PTC-100 thermal cycler (MJ Research, South San Francisco, CA), with 35 cycles per sample. Cycling temperatures were as follows: denaturing 94°C; annealing, 58°C; and extension, 70°C. The following primers were used to isolate the TCF binding region within the TGF-β3 gene promoter: forward, 5′-CCGAGGTGCTGGTGACCCTG-3′ and reverse, 5′-CCAGTGAGTAGGTGGGGAGA-3′. Samples were run on 1.5% agarose gels containing ethidium bromide (1.5 μl) and visualized with a ChemiDoc XRS Imager (Bio-Rad, Hercules, CA).
Plasmid Construction and Site-directed Mutagenesis
A recombinant plasmid was made by inserting the −1-kb fragment of the human TGF-β3 gene promoter into the pGL3 luciferase plasmid vector (Promega, Madison, Wi). The insert was amplified by PCR using a human genomic DNA template with addition of KpnI and HindIII restriction sites to primers matching those found in the pGL3 vector. The following primers (Integrated DNA Technologies) were used: forward, 5′-TTGGTACCCCAAGGGAATGAGCGAGAGA3′ and reverse, 5′-CCCAAGCTTGTGTGAGCTGGGAAGAGAGG-3′. Restriction digest with KpnI and HindIII enzymes (New England Biolabs, Ispwich, MA) was performed for both the insert and vector, followed by ligation with T4 DNA ligase (New England Biolabs). Two base-pair mutagenesis PCR within the TCF binding region was performed using the following primer sequences: forward, 5′-GAGGCAGCATGAACGACGTCATTTAGAAAG-3′ and reverse, 5′-CCTTCTAAATGACGTCGTTCACGCTGCCTC-3′. Mutagenesis caused formation of a unique AatII restriction site within the recombinant plasmid. The sequence changes for the −704 bp to −688 bp TCF binding site were as follows: wild type, 5′-GCGTGAACAAAGTCATT-3′; and mutant, 5′-GCGTGAACGACGTCATT-3′. Mutagenesis was confirmed by restriction digest with AatII and HindIII enzymes (New England Biolabs).
Luciferase Reporter Gene Assays
Luciferase reporter gene assays were conducted using the Luciferase Assay System (Promega) and its corresponding protocol. All plasmids (500 ng) were transfected into cells using Lipofectamine and Plus reagents (Invitrogen) according to the manufacturer's guidelines. Light units were measured with a Luminometer TD-20/20 (Turner Designs, Sunnyvale, CA). Assays were normalized for transfection efficiency by cotransfecting cells with a β-gal control plasmid and were detected with the Luminescent β-gal control assay kit (Clontech, Mountain View, CA). Experimental (Luciferase) results were divided by the β-gal results to provide normalized data. The pTOPFLASH-Lux reporter construct was provided by Dr. Hans Clevers (Netherlands Institute for Developmental Biology, Utrecht, The Netherlands). The p3TP-Lux reporter plasmid was provided by Dr. Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY).
Invasion and migration were assessed using the Innocyte Cell Migration Assay kit (EMD Biosciences, San Diego, CA). Transwell plates (96-well) containing 8-μm pores were coated with 250 ng/ml type I collagen (BD Biosciences, San Jose, CA) and 100 ng/ml Laminin (Invitrogen). Cells migrated toward 10% serum into the lower chambers. Migrated cells in the lower chamber were stained with a Calcein-acetoxymethyl ester fluorescent dye. Excitation max (485 nm)/emission max (520 nm) was assessed using a standard fluorescent plate reader (BD FACSArray bioanalyzer; BD Biosciences).