All cells used in the present study were from the American Type Culture Collection (Manassas, VA). The cell media and culture conditions were previously described (Shirakihara et al, 2007
). NMuMG cells were cultured for several days and passaged every 2 days in medium with or without 1 ng/ml TGF-β1, 30 ng/ml FGF-2, and 100 μg/ml heparin. All cells were grown in a 5% CO2
atmosphere at 37°C.
Reagents and antibodies
Recombinant human TGF-β1, FGF basic (FGF-2), and FGF-7 were purchased from R&D Systems (Minneapolis, MN). SU5402, PI-103, U73122, and GM6001 were purchased from Calbiochem (Darmstadt, Germany). U0126 was obtained from Promega (Madison, WI). The mouse monoclonal anti-α-tubulin and anti-α-SMA antibodies were purchased from Sigma-Aldrich. The mouse monoclonal anti-Erk2 antibody was from Millipore (Bedford, MA). The rabbit polyclonal anti-phospho-Erk1/2 antibody was obtained from Cell Signaling (Beverly, MA). The rabbit polyclonal anti-δEF1 antibody was from Santa Cruz Biotechnology (Lot No. E1408, Santa Cruz, CA), and the mouse monoclonal anti-CtBP1 and anti-E-cadherin antibodies were from BD Transduction Laboratory (Lexington, KY). Rabbit monoclonal anti-calponin antibody was from Abcam (Cambridge, UK). Tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin and propidium iodide (PI) were from Sigma-Aldrich (Saint Louis, MO) and Molecular Probes (Eugene, OR), respectively.
Immunoprecipitation, immunoblotting, and immunofluorescence labelling
The procedures used for immunoprecipitation, immunoblotting, and immunofluorescence were previously described (Shirakihara et al, 2007
). Immunodetection was performed with the ECL blotting system (Amersham Bioscience, Piscataway, NJ) and Luminescent Image Analyzer (LAS4000, Fujifilm, Tokyo, Japan). Fluorescence was examined using a confocal laser scanning microscope (Carl Zeiss, Thornwood, NY).
siRNAs were transfected into cells according to the protocol recommended for HiPerFect reagent (Qiagen, Valencia, CA). NMuMG cells were transiently transfected with siRNAs against mouse δEF1 (Stealth RNAi; Invitrogen, Carlsbad, CA), mouse SIP1 (GGAAAAACGUGGUGAACUA; B-Bridge, Sunnyvale, CA), or mouse CtBP1 (Stealth RNAi; Invitrogen). The final concentrations of the siRNAs were 5 nM, except for SIP1 siRNA, which had a final concentration of 10 nM.
RNA extraction and RT–PCR
Total RNA was extracted and analysed by quantitative RT–PCR analysis as previously described (Shirakihara et al, 2007
). The specificity of the detected signals was confirmed by a dissociation curve, which consisted of a single peak. Values were normalized to the TATA-binding protein (TBP). Conventional RT–PCR analyses were performed using the PC-708 Program Temp Control System (ASTEC, Fukuoka, Japan). The RT–PCR primer sequences are listed in Supplementary Table S1
Cell motility assay
NMuMG cells were treated with TGF-β, FGF-2, or both for 4 days and seeded in six-well tissue culture plates. The cell monolayers were wounded by scratching with sterile plastic 200 μl micropipette tips and photographed using phase-contrast microscopy immediately and 12 h after wounding. The assays were independently performed in triplicate. The migration distance of each cell was measured after the photographs were converted to Photoshop files (Adobe, San Jose, CA).
NMuMG cells were treated with TGF-β or both TGF-β and FGF-2 for 4 days and seeded onto Cell Culture Inserts (8 μm pore size; BD Falcon, Franklin Lakes, NJ) coated with Type IV collagen (BD Biosciences, San Jose, CA). After 24 h, the cells that had not invaded into the lower surface of the filters were removed from the upper face of the filters using cotton swabs. The cells that had invaded into the lower surface of the filters were fixed in methanol and stained with Giemsa. Invasion was quantitated by visually counting the photographed cells.
Collagen gel contraction assay
Type I collagen gel was prepared using an 8:1:1 ratio of cold collagen solution (Cellmatrix I-P; Nitta Gelatin, Osaka, Japan), 10 × concentrated MEM (Invitrogen), and collagen dilution buffer containing 0.05 N NaOH, 2.2% NaHCO3, and 200 mM HEPES, pH 7.4. NMuMG cell suspensions (2.0 × 105 cells/200 μl) were mixed in 800 μl of the collagen gel solution. A measure of 1 ml of the mixture was added to each well of 12-well culture plates and allowed to solidify at 37°C for 30 min. After solidification, 1 ml of DMEM containing 4.5 g/l glucose, 10% FBS, 10 μg/ml insulin, 50 U/ml penicillin, and 50 μg/ml streptomycin was overlaid to float the gel. The floating gels were incubated at 37°C in 5% CO2 for 2 days. The gel surface area was quantified based on pixel number using ImageJ (US National Institutes of Health, Bethesda, MD). The relative changes in the surface area are reported as the percentage of the original surface area.
NMuMG cells were treated with TGF-β, FGF-2, or both for 4 days. Then, the cells were cultured with serum-free media for 24 h, and the conditioned media were collected. Equal amounts of samples were mixed with 3 × non-reducing SDS–PAGE sample buffer. The samples were applied to a 10% polyacrylamide gel impregnated with 1 mg/ml gelatin (Sigma). After running, SDS was removed from the gel by washing three times with 2.5% Triton X-100 solution for 20 min. Then, the gels were incubated overnight at 37°C in buffer containing 50 mM Tris–HCl, pH 7.6, 5 mM CaCl2, and 200 mM NaCl. The gel was stained with 0.5% Coomassie blue R250 (CBB) in 50% methanol and 5% acetic acid for 2 h, and subsequently destained with a solution containing 40% methanol and 10% acetic acid.
In vitro three-dimensional invasion assay
A mixture of collagen I (Koken, Tokyo, Japan) and DMEM containing 10% FBS, 10 mM HEPES, and 0.1% sodium bicarbonate were added to 12-well culture plates. The collagen gel was allowed to solidify at 37°C for 1 h. The final concentration of collagen gel was 2.4 mg/ml. MCF-7 cells stably expressing Ha-ras G12V were infected with lentivirus encoding green fluorescent protein (MCF-7-GFP). NMuMG cells were pretreated with TGF-β or TGF-β and FGF-2 for 2 days and then mixed with MCF-7-GFP cells and seeded on collagen gels. The culture media containing each of the noted ligands was changed every 2 days. After 1 week, the gels were fixed with Mildform (Wako, Osaka, Japan), embedded in paraffin, and stained with haematoxylin–eosin (HE). The same sections were used to detect GFP by confocal laser scanning microscopy.
Online supplemental material