CACGT-39) or cultured in the presence of transfection reagent alone, served as negative controls. The concentration of siRNAs used was selected based on previous studies of primary EVT cultures [20]. Following optimization of the TransMessenger:- siRNA concentration ratio, all experiments were performed for at least 24 h. Expression Vectors Mammalian expression vectors (pcDNA3.1; Invitrogen, Carls- bad, CA) containing either a full length human ADAMTS-12 cDNA (pcDNA3-ADAM-TS12-HA) or a full-length human ADAMTS-12 cDNA in which the catalytic domain had been inactivated by site directed mutagenesis (pcDNA3-ADAM-TS12- MUT) were generously provided by Dr. S. Cal (Universidad de Oviedo, Spain) herein abbreviated as A12FL and A12Mut, respectively. These constructs have been described [21]. A12Mut cDNA construct has two point mutations in the metalloproteinase domain, where the thymine (465) and adenine (466) have been substituted for adenine and cytosine resulting in the translated protein consisting of a glucine amino acid instead of histidine thus rendering the protein catalytically inactive. A full- length human ADAMTS-1 cDNA (Genbank Accession No. NM006988) was purchased from Origene (Rockville, MD, USA) and cloned into pcDNA3.1. A clone (A1) containing the ADAMTS-1 cDNA in the forward orientation was subsequently identified by DNA sequencing. A pcDNA3.1 expression vector containing the b-galactosidase gene (pcDNA3-LacZ; Invitrogen) was used to determine transfection efficiency and served as a control for these studies. Generation of stably transfected JEG-3 cell lines Stable transfections were performed to establish clonal JEG-3 cell lines constitutively expressing A12FL, A12Mut, A1 or LacZ. Each expression vector (1.0 mg/ml) was transfected using Exgen 500 transfection reagent (Fermentas, Burlington, ON, Canada) according to the manufacturer’s protocol. Selection began 48 h post transfection using 400 mg/ml G418 in DMEM, and subcolonies were isolated by limiting dilution and expanded into cell lines maintained in the selection medium. At least three independent clones were selected per construct and protein expression confirmed by Western blotting (data not shown). Transwell invasion assays Cell invasion assays were performed using Transwells fitted with Millipore Corp. membranes coated with a thin layer of growth factor-reduced Matrigel (6.5-mm filters, 8-mm pore size) as described [22]. Briefly, 26104 cells/200 ml of DMEM supple- mented with 1% FBS were plated in the upper chambers and cultured for 24 h (EVTs) or 48 h (JEG-3 cells). Cells from the upper surface of the Matrigel layer were removed by gentle swabbing, while transmigrated cells attached to the membrane were fixed in 4% paraformaldehyde and stained with eosin. The filters were rinsed with water, excised from the Transwells, and mounted upside-down onto glass slides. Invasion indices were determined by counting the number of stained cells in 10 randomly selected, non-overlapping fields at 4006 magnification using a light microscope. Cell invasion was tested in triplicate wells, on three independent occasions. A subset of these experiments were performed in the presence of a synthetic RGD (Arg-Gly-Asp) or control peptide (1 mM; BIOMOL International, Plymouth Meeting, PA) following procedures of Buckley et al [23], a bicyclic RGD peptide (H-Glu[cyclo(Arg-Gly-Asp-D-Phe-Lys)]2; Peptides International) shown to possess high affinity towards avb3 integrin (IC50 = 0.9 nM) with low affinity for avb5 and aIIBb3 ADAMTS-12 and Trophoblast Invasion integrins (IC50 = 10 nM) and a cyclo (Arg-Ala-Asp-D-Phe-cys; Peptides International) peptide used as a negative control. In addition to the RGD peptides, a monoclonal function perturbing av integrin antibody (Chemicon International; mouse anti-integrin av antibody; clone AV1) was used in Transwell invasion experiments [24]. Prior to seeding cells into invasion chambers, 5.46105 cells in media suspension were pre-incubated in the presence of the indicated peptide/antibody for 30 min. Cell aggregation assays Cell aggregation assays were performed using the cell hanging- drop method [25]. Briefly, trypsinized single cell suspensions (1.56105 cells/ml) treated with EDTA and passaged through 20 mm nylon sieves were prepared in DMEM media containing 10% FBS. From these suspensions, three 20 ml droplets were pippetted onto the underside of 6 cm culture dish lids, which were carefully inverted and placed onto its bottom well containing 2 ml DMEM media and cultured for 2 or 8 h. After incubation, culture dish lids were again inverted and glass coverslips were mounted onto the cell-drop suspensions. The extent of cellular aggregation was quantified by counting cell aggregates under a light microscope fitted with a digital camera (2006 magnification); aggregates were allocated into 3 groups: ,5 cells/aggregate, 5–15 cells/aggregate, and .15 cells/aggregate. Each experiment was repeated a minimum of three times. Native ECM-binding assay Stably transfected JEG-3 cells were plated in 24-wells in triplicate at high density and allowed to grow to near confluency for 24 h. Cell media was replaced with fresh DMEM media and cultured for an additional 48 h to allow for matrix deposition. Cell monolayers were removed by incubation with 20 mM NH4OH (265 min) according to Gospodarowicz et al [26] followed by washing in 16 PBS. JEG-3 cells growing in parallel were trypsinized, passaged through a 40 mm nylon sieve, plated onto the ECM deposited by the transfected JEG-3 cell lines and cultured for 30 min. Cells were washed vigorously in 16PBS, fixed in 4% paraformaldehyde and stained with eosin. ECM binding ability was determined by counting the number of cells that had adhered to the deposited ECM under a microscope. ECM Cell Adhesion Assay A fluorimetric ECM cell adhesion array kit (Chemicon International) allowed us to screen for and quantitate the binding affinity of cells to 7 different human ECM proteins. Collagen I, collagen II, collagen IV, fibronectin, laminin, tenascin, and vitronectin were pre-coated onto 96-well microtiter plates arranged in 1268-well strips. In each 8-well strip, one BSA- coated well served as a negative control. Following the manufacturers instructions, we seeded 16105 cells suspended in DMEM (100 ml) onto the coated ECM-substrates and analysed for the cells ability to bind to the respective ECM proteins using a fluorescence plate reader (485/530 nm). The plating of tropho- blastic cells onto 8-well ECM protein coated strips was performed on three independent occasions (n = 3). Alpha and Beta Integrin Binding Assay A fluorimetric a/b Integrin-Mediated Cell Adhesion Array Combo Kit (Chemicon International) was used to assess the integrin repertoire expressed on the cell surface. The kit is comprised of two 96-well plates: an a-integrin binding 96-well plate and a b-integrin 96-well plate. Each kit uses mouse monoclonal antibodies generated against human a (a1, a2, a3, PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e18473