Supplementary MaterialsSupplementary Data. decidual (= 4) were used to examine ADAM8 localization and expression by RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cellCmatrix binding was tested using fibronectin-adhesion assays, and ADAM8-1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation FLJ31945 experiments and function-promoting/inhibiting antibodies. MAIN RESULTS AND THE ROLE OF CHANCE Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G+ trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic Mometasone furoate cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cellCmatrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates 1-integrin activation and promotes cell migration through a mechanism dependent on 1-integrin function. LIMITATIONS, REASONS FOR CAUTION The primary limitation of this study was the use of experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Mometasone furoate Further, patient information corresponding to tissues obtained by elective terminations was not available. WIDER IMPLICATIONS OF THE FINDINGS The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental Mometasone furoate development. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant) and the Canadian Institutes of Health Research (CIHR-Open Operating Grant). There are no conflicts or competing interests. TRIAL REGISTRATION NUMBER NA. expression of the MHC class-I molecule, HLA-G, the up-regulation of specific integrin cellCmatrix adhesion proteins (i.e. 5 integrin), and production of proteases important in extracellular matrix and cell membrane remodeling (Davies = 24) and decidual tissues (= 4) were obtained from women (19C35 years of age) providing written informed consent undergoing elective terminations of pregnancy at British Columbias Womens Hospital, Vancouver, Canada. All samples were confirmed to have come from viable pregnancies by ultrasound-measured fetal heartbeat. Ethical approval The use of these tissues was approved by the Research Ethics Board on the use of human subjects, University of British Columbia (H13-00 640). FACS purification of placental cells Placental villi single cell suspensions were generated from fresh first trimester placental specimens (= 4) by enzymatic digestion and analyzed by flow cytometry following protocols adapted from Beristain (2015) and Aghababaei (2015). Briefly, placental villi were digested for 1 h at 37C in Hanks Balanced Salt Solution (HBSS), 750 U/ml collagenase and 250 U/ml hyaluronidase. Organoids obtained after vortexing were subjected to red blood cell lysis in 0.8% (w/v) NH4Cl, further dissociation in 0.25% (w/v) trypsin for 2 min, 5 mg/ml dispase with 0.1 mg/ml DNase I for 2 min, and filtered through a 40 m mesh to obtain single cells. Contaminating immune cells were removed from the cell admixture by EasySep immuno-magnetic bead purification Mometasone furoate (all reagents obtained from StemCell Technologies, Vancouver, Canada). Following magnetic bead exclusion, 2.5 106 cells were blocked with Fc receptor antibody (eBioscience, San Diego, CA, USA), and incubated with the following antibodies on ice for 30 min: anti-CD45 (clone 2D1, eBioscience), anti-CD49f-PE-Cy7 (clone GoH3, eBioscience) and anti-HLA-G-PE (clone 87 G, eBioscience). Dead cells were excluded from analysis by staining with 7AAD (eBioscience). The cell surface markers CD49f and HLA-G were used to identify placental trophoblast cell populations, while CD45 was used to identify and exclude contaminating immune cells that were not completely removed by EasySep magnet purification (StemCell Technologies). FACS analysis was performed using FACSDiva (BD, San Diego, CA, USA) and FlowJo software (Tree Star, Inc., Ashland, USA). Cell sorting was performed on a FACSAria (BD) into tubes containing ice-cold HBSS/2% fetal bovine serum (FBS). Magnetic bead purification of placental cells Placental villi single cell Mometasone furoate suspensions were obtained from first trimester placental villi by enzymatic digestion, density gradient centrifugation.