in cultures of invasive EVTs propagated from these tissues and in the poorly invasive JEG-3 choriocarcinoma cell line. Of these, only ADAMTS-12 was found to be present in EVTs at significantly higher levels than in JEG-3 cells. The ability of transforming growth factor (TGF)-b1 and interleukin (IL)-1b, two cytokines assigned counter-regulatory roles in human placentation [3,15], to differen- tially regulate ADAMTS-12 mRNA levels in cultures of EVTs supported our hypothesis that this ADAMTS subtype confers an invasive phenotype on human trophoblastic cells. Loss- or gain-of- function studies subsequently confirmed that this ADAMTS subtype, independent of its intrinsic proteolytic activity, plays an active and dominant role in human trophoblastic cell invasion in vitro through a mechanism controlling cell-ECM interaction. Specifically, ADAMTS-12 modulated cell invasion by regulating the expression and function of the avb3 integrin heterodimer. Materials and Methods Tissues Samples of first trimester placental tissues were obtained from women undergoing elective termination of pregnancy (gestational ages ranging from 6–12 weeks). The use of these tissues was approved by the Committee for Ethical Review of Research on the use of human subjects, University of British Columbia. All women provided informed written consent. Tissue culture EVT cultures were propagated from first trimester placental explants essentially as described [16]. The purity of the EVT cultures was determined by immunostaining for human cytoker- atin filaments 8 and 18. Only cultures that exhibited 100% immunostaining for these cytokeratins were included. 56106 EVTs were grown to 80% confluency and treated with TGF-b1 (0.1–10 ng/ml) or IL-1b (1–1000 IU/ml) for 24 h, or TGF-b1 (5 ng/ml) or IL-1b (100 IU/ml) for 0–48 h. EVTs cultured in vehicle (ethanol) served as controls. Specific cultures were subjected to a function-perturbing monoclonal antibody against human TGF-b1 (Sigma Aldrich; 10 mg/ml; clone 9016.2) or IL-1b (Sigma Aldrich;100 IU/ml; clone 8516.311) for 24 h. JEG-3 trophoblastic cell were purchased from ATCC, Manassas, VA, USA. On-going cultures were maintained in DMEM containing 25 mM glucose, L-glutamine, antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin) and supplemented with 10% FBS. Primer Design and preparation of cDNA Probes Primer sets for ADAMTS-1 through -12 [17] or GAPDH were synthesized at the NAPS Unit, UBC. The nucleotide sequences of primers, optimized PCR conditions, and the sizes of the PCR products are listed in Table S1. To generate cDNA probes for each ADAMTS or GAPDH, PCR products were generated from human placental tissue, subcloned into the PCR II vector and confirmed by nucleotide sequencing. A second set of ADAMTS- 12-specific primers, in which a stretch of nucleotides correspond- ing to a sequence present within the target ADAMTS-12 PCR product was incorporated into the 39-end of the forward primer; These were used for quantitative competitive (QC)-PCR analysis of ADAMTS-12 mRNA levels in cultures treated with TGF-b1 and IL-1b. This follows a similar approach as reported for examining urokinase plasminogen activator/plasminogen activa- tor inhibitor-1 and MMP/TIMP mRNA levels [18]. Semiquantitative PCR and Southern blot analysis Total RNA was prepared from tissue samples or cells using an RNeasy Mini Kit (Qiagen, Inc, CA) following the manufacturer ADAMTS-12 and Trophoblast Invasion instructions. Aliquots (,1 mg) of the total RNA extracts were then reverse-transcribed into cDNA using a First Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Oakville, ON, Canada). Semiquantitative PCR was performed using the primer sets listed in Table S1. All PCR reactions were performed on 3 separate occasions (n = 3). PCR products were separated by standard electrophoresis followed by Southern blotting according to the methods of MacCalman et al [19]. The autoradiograms were scanned using a laser densitometer (Scion Corporation, Frederick, MD, USA) and the absorbance values of the distinct ADAMTS PCR products normalized relative to the corresponding GAPDH value. QC-PCR The QC-PCR strategy employed in these studies is based upon the competitive co-amplification of a known amount of compet- itive ADAMTS-12 PCR product added to aliquots of first strand cDNA prepared from our primary cultures of EVTs. The PCR conditions were: 1 min at 94uC, 1 min at 58.5uC and 1.5 mins at 72uC for 28 cycles followed by a final extension at 72uC for 15 min. The resultant target and competitive ADAMTS-12 PCR products were separated using gel electrophoresis. PCR products identity was confirmed by subcloning and DNA sequencing (data not shown). To determine the optimal amount of competitive cDNA to be added to each reaction, PCR was performed using either a fixed amount of template cDNA combined with decreasing concentrations of competitive cDNA or conversely, a fixed concentration of competitive cDNA combined with decreas- ing amounts of template cDNA. The intensity of ethidium bromide staining of the PCR products was analysed by UV densitometry, and volume counts determined using the Scion Image computer software. The competitive ADAMTS-12 cDNA was subsequently added to each reaction at a concentration of 4.8861023 pg/ml. Cell lysis, immunoprecipitation and immunoblot analysis Cultures of JEG-3 cells or EVTs were washed in PBS and incubated in cell extraction buffer (Biosource International, Camarillo, CA) supplemented with 1.0 mM PMSF and Complete Mini proteinase-inhibitor cocktail (Roche) for 30 minutes. Protein concentrations were determined using a BCA kit (Pierce Chemicals, Rockford, IL, USA). Lysates (500 mg) were immuno- precipitated with an av integrin antibody (Chemicon Internation- al) overnight at 4uC followed by incubation with protein G plus protein A agarose beads (Invitrogen) for 1 hr. The precipitated protein complexes were washed at 4uC in RIPA buffer without sodium deoxycholate or SDS. For immunoblotting, 30 mg of cell protein lysate was resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed using polyclonal antibodies against the carboxyl terminal of human ADAMTS-12 (Santa Cruz Inc, Santa Cruz, CA, USA) and ADAMTS-1 (AbCAM), and monoclonal antibodies directed against av, b1, b3 integrin subunits (Chemicon International), phospho-397 FAK and FAK (Cell Signaling). The blots were reprobed with a monoclonal antibody directed against human b- actin (Sigma Aldrich, St, Louis, MO) to assess protein loading. siRNA transfection siRNA (Xeragon Inc, Germantown, MD; 13.5 mg/100 mm2 culture dish) targeting the human ADAMTS-12 mRNA transcript (59-AAGCCCGTCCCTCCACCTACA-39) was transfected into EVTs using TransMessenger transfection reagent (Qiagen) according to manufacturer’s protocol. EVTs transfected with a non-silencing, scrambled siRNA (59- ATTTCTCCGAACGTGT- PLoS ONE | www.plosone.org 2 April 2011 | Volume 6 | Issue 4 | e18473