Recombinant human jagged1 fc chimera protein

Bioflex culture plates (untreated, Flexcell Int.) were coated with 2.2 µg/cm² of bovine fibronectin (Thermo Fisher Scientific) on 4 cm² of the center of the wells for 1 h at 37°C. The wells were treated with 1% Pluronic F-127 (Sigma-Aldrich) for 5 min at room temperature to prevent aspecific adhesions. For the induction of Notch signaling, 50 μg/ml Recombinant Protein G (Thermo Fisher Scientific) in PBS were added to the fibronectin-coated membranes and incubated overnight at room temperature. Plates were incubated with 2 μg/ml of Recombinant Human Jagged1-Fc Chimera Protein (R and D Systems) in 0.1% bovine serum albumin (BSA)/PBS for 3 h at room temperature. Cells were immediately seeded on Bioflex culture plates with densities of 100.000 cells/4 cm² for synthetic VSMCs and 150.000 cells/4 cm² for contractile VSMCs and left to attach overnight. Notch inhibition was performed by adding 10 μg/ml γ-secretase inhibitor DAPT (Sigma- Aldrich) to the culture media from stock solutions in sterile dimethyl sulfoxide (DMSO) (Sigma-Aldrich). The same concentration of the vehicle DMSO was added to the other samples for comparison. DMSO/DAPT was refreshed every 24 h.

Paraquat dichloride hydrate, DAPT (N-(N-(3,5-difluorophenyl)-L-alanyl)-S-phenylglycine t-butylester) and SB431542 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human Jagged-1 Fc chimera protein was manufactured by R&D system (Minneapolis, MN, USA). Antibodies against NADPH, β-actin, TGF-β1 were purchased from Abcam (Cambridge, UK). Antibody against α-SMA and E-cadherin are provided by Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against Jagged-1, Notch1-4, Smad3 and phospho-Smad3 were purchased from Cell Signaling (Beverly, MA, USA).

Hepatocytes were isolated from the murine liver using the collagenase perfusion method^{8 (link)} and subjected to primary culture on a bovine type I collagen-coated dish (AGC TECHNO GLASS, Shizuoka, Japan). In some experiments, primary hepatocytes were cultured in dishes precoated with recombinant human Jagged-1 Fc-chimera protein (R&D Systems, Minneapolis, MN). Gamma secretase inhibitor IX (DAPT; Merck, Darmstadt, Germany) was diluted in dimethyl sulfoxide (Sigma-Aldrich) before use.