Human mammary epithelial (HME) cells were immortalized with human papilloma virus E6/E7 and were characterized as being positive for keratin 19 [6
]. Cells were cultured in Ham's F-12 medium supplemented with 5% fetal bovine serum (FBS), hydrocortisone (1 μg/ml), insulin (5 μg/ml), epidermal growth factor (EGF; 10 ng/ml), cholera toxin (100 μg/ml), fungizone (2.5 μg/ml) and gentamycin (5 μg/ml), at 37°C under 10% CO2
Generation of stable hairpin short interfering RNA-CCN6 HME cells
Hairpin short interfering RNA (siRNA) 5'-GATCCCGCCAGGGGAAATCTGCAATGTTCAAGAGACATTGCAGATTTCCCCTGGTTTTTTGGAAA-3' and the complementary strand 5'-AGCTTTTCCAAAAAACCAGGGGAAATCTGCAATGTCTCTTGAACATTGCAGATTTCCCCTGGCGG-3' were synthesized (Invitrogen, Carlsbad, CA, USA), and annealed hairpin siRNA-CCN6 inserts were cloned into pSilencer2.1-U6 hygro expression vector (Ambion, Austin, TX, USA). The sequence of siRNA-CCN6 (insert in) expression vector was confirmed by sequencing (University of Michigan DNA Sequencing Core). HME cells were transfected with pSilencer2.1-U6 negative control (siRNA-control; Ambion) or siRNA-CCN6 plasmids by using FuGene 6 transfection reagent (Roche-Boehringer Mannheim, Mannheim, Germany). Stable transfectants were established by culturing transfected cells in the described medium supplemented with 100 μg/ml hygromycin (Invitrogen) for 4 weeks.
Total RNA was isolated from HME cells and siRNA HME clones with a Trizol kit (Life Technologies, Inc., Gaithersburg, MD, USA). First-strand cDNA synthesis was performed by using 1 μg of total RNA with AMV reverse transcriptase (Promega, Madison, WI, USA) and oligo(dT) as a primer. A 2 μl portion of the reaction mixture was used for amplification by PCR. The following primers were used: CCN6 forward primer 5'-ATGCAGGGGCTCCTCTTCTCC-3' and CCN6 reverse primer 5'-CTTGAGCTCAGAAAATATATC-3' to amplify a 1,050 bp product; E-cadherin forward primer 5'-CCTTCCTCCCAATACATCTCCC-3' and E-cadherin reverse primer 5'-TCTCCGCCTCCTTCTTCATC-3' to amplify a 600 bp product. PCR was performed under the following conditions: denaturing for 1 minute at 94°C, annealing for 1 minute at 58°C for CCN6 and at 65°C for E-cadherin, and elongation for 1 minute at 72°C, for 35 cycles.
Cells were grown on glass coverslips, fixed with methanol for 5 minutes at -20°C and then permeabilized with 0.2% Triton X-100 for 15 minutes at room temperature 25°C. The coverslips were then saturated for 30 minutes with 2% bovine serum albumin in PBS. After being washed in PBS, cells were incubated for 1 hour with a monoclonal antibody against E-cadherin (dilution 1:50, BD Transduction Laboratories, San Jose, CA, USA). Cells were exposed for 1 hour to Alexa Fluor anti-mouse Ig G (Molecular Probes, Eugene, OR, USA). After being washed with PBS three times, the coverslips were then mounted with prolong Gold anti-fade reagent with 4',6-diamidino-2-phenylindole and the borders were sealed with transparent nail polish.
For studies of anchorage-independent growth we performed soft agar assays on stable clones of HME/CCN6 siRNA and on HME control cells. Each well of a six-well plate was first layered with 0.6% agar diluted with 5% FBS-supplemented Ham's F-12 medium complete with growth factors. The cell layer was then prepared by diluting agarose to concentrations of 0.3% with 5 × 103 cells in 2.5% FBS-supplemented Ham's F-12 (1.5 ml per well). Plates were maintained for 3 weeks at 37°C under 10% CO2. Colonies were counted under a microscope with a grid. The experiment was performed three times independently.
Basement membrane matrix invasion
HME/CCN6 siRNA and HME controls were trypsinized and 300 μl of 106 cells/ml in serum-free medium were seeded at equal numbers onto the extracellular matrix layer (ECM; Chemicon, Temecula, CA, USA). Subsequently, we added 500 μl of medium containing 5% FBS to the lower chamber. After incubation for 24 hours, the non-invading cells and ECM gel from the interior of the insert were removed gently with a cotton swab. The invasive cells on the lower surface of membrane were stained, air dried, and photographed. The invaded cells were counted under the microscope. The experiment was performed three times independently.
Random cell motility was determined as described in the motility assay kit (Cellomics Inc., Pittsburg, PA, USA). Cells were harvested and 500 cells per well were suspended in 2.5% FBS medium and plated on top of a field of microscopic fluorescent beads in a 96-well plate. After incubation for 16 hours, cells were fixed and areas of clearing in the fluorescent bead field corresponding to phagokinetic cell tracks were quantified with an NIH ScionImager. The experiment was performed three times independently.
HME cells and HME/CCN6 siRNA cells were serum-starved for 24 hours and subsequently stimulated for 15 minutes with 25 ng/ml IGF-1. Proteins were obtained by lysing the cells in a buffer composed of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride, 1 μg/ml leupeptin, and 10 μg/ml aprotinin. The IGF-1R was immunoprecipitated overnight from 500 μg of cell (protein) lysate with anti-IGF-1R monoclonal antibody (Oncogene, San Diego, CA, USA) at 4°C. After isolation with protein A/G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the immunocomplexes were separated by 7.5% SDS-PAGE. The proteins were transferred to a poly(vinylidene difluoride) membrane and subsequently detected by immunoblotting with anti-IGF-1R β subunit polyclonal antibody (Santa Cruz Biotechnology). Tyrosine phosphorylation of immunoprecipitated IGF-1R was assessed with anti-phosphotyrosine monoclonal antibody PY20 (Transduction Laboratories, Lexington, KY, USA).
Western blot analysis
Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40, 1 mM Na3VO4, 1 mM phenylmethylsulphonyl fluoride, 1 μg/ml leupeptin, and 10 μg/ml aprotinin). Cell lysates (50 μg of protein) were fractionated by SDS-PAGE and transferred to Immobilon-P membrane (Millipore, Billerica, MA, USA). The membranes were blocked for 1 hour with Tris-buffered saline containing 5% non-fat milk and 0.1% Tween 20. Immobilized proteins were probed with antibodies specific for phosphorylated and total insulin receptor substrate-1 (IRS-1; Upstate Biotechnology), E-cadherin and vimentin monoclonal antibodies (BD Transduction Laboratories) and antibodies against cytokeratin 18 (c-04, ab668, Abcam Inc, Cambridge MA, USA). CCN6 was detected with a polyclonal anti-CCN6 antibody developed against an immunogenic peptide Ac-PEGRPGEVSDAPQRKQ-CONH2 corresponding to amino acids 31 to 46 of human CCN6 with the assistance of Covance.
IGF-1 growth assay
HME/CCN6 siRNA and HME control cells were plated in 96-well plates at a concentration of 5 × 104 cells/ml and serum-starved for 24 hours. Subsequently, 25 ng/ml human recombinant IGF-1 (Upstate Biotechnology) was added. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reagents were added 24 hours later in accordance with the manufacturer's protocol, and the plate was read at a wavelength of 595 nm. The experiment was performed with triplicate samples.
HME/CCN6 siRNA and HME control cells were plated at a concentration of 5 × 104 cells/ml and serum-starved for 24 hours. Subsequently, 25 ng/ml human recombinant IGF-1 (Upstate Biotechnology) was added. Serum-starved and IGF-1-treated HME control cells and HME/CCN6 siRNA cells were pulsed for 2 hours with 10 μM bromodeoxyuridine (BrdU; Roche, Indianapolis, IN, USA), harvested with trypsin-EDTA, and fixed with 70% ethanol at -20°C for at least 1 hour. Cells were resuspended in 2 M HCl for 20 minutes at room temperature. After centrifugation, the cell pellets were resuspended in borax (pH 8.5) to neutralize any residual acid. After brief centrifugation the pellets were washed in 1 ml PBS and resuspended for 1 hour in fluorescein isothiocyanate-labeled anti-BrdU antibody (catalogue no. 347583; Becton Dickinson San Jose, CA, USA) in the dark. Cells were washed in PBS, resuspended in PBS containing propidium iodide, and analyzed for propidium iodide and BrdU staining on a Becton-Dickinson flow cytometer with the use of Cellquest software. The experiment was performed three times independently.