Jag1 fc

Synovial fibroblasts derived from RA synovial tissue were cultured in DMEM 10% fetal bovine serum as previously described ³⁸ . For fibroblast Notch activation experiments, cell culture plates were coated with recombinant DLL4 or JAG1-FC (R&D systems) overnight at 4 degrees, at 5 μg/ml or at the concentration indicated for each experiment. Fibroblasts were then seeded on DLL4-, JAG1-FC- , or vehicle-coated plates for 72 to 120 hours. For cytokines and growth factors stimulation experiments, recombinant proteins were purchased (R&D systems) and reconstituted in DMSO or PBS per manufacturer’s instructions. Recombinant protein were diluted in media and then added to cultured fibroblasts at the following concentrations: TGFB1 10 ng/ml, BMP7 10 ng/ml, ACTIVIN A 10 ng/ml, WNT3A 100 ng/ml, WNT5A 100 ng/ml, EGF10 ng/ml, TNF 10 ng/ml, IFN-gamma or PDGF-BB 50 ng/ml. For flow cytometric analysis of Notch pathway in fibroblast-endothelial cell co-culture experiments, HUVECs (Lonza), passage 3 – 7, were cultured in a 1:1 ratio with synovial fibroblasts in EGM2 media for 72–96 hours in the presence or absence of 10 μM DAPT as indicated. For siRNA experiment, all siRNAs (Silencer Select) were purchased from Life Technologies. Fibroblasts were transfected with siRNA by reverse transfection at 30 nM using RNAi Max reagent (Life Technologies) as previously described ³⁸ .

24-well tissue culture plates were coated with Dll1-Fc (R&D Systems, 3970-DL-050), Dll4-Fc (Sino Biological, 10171-H02H-50) or Jag1-Fc (R&D Systems, 599-JG-100) (4 μg/mL per well for each ligand) in PBS for 2 hours at room temperature. NIH3T3 cells (0.5 × 10⁵ cells/well) were plated in ligand-coated wells and incubated overnight. Transfection of control and Notch reporter constructs, media changes, β-galactosidase, and luciferase reporter assays were carried out, as above. Three biological replicates were performed for each condition (n=3).