Htgf β1

Human LR-MSCs were treated with or without hTGF-β1 (Cell Signaling Technology, Beverly, MA) at 10 ng/ml for indicated periods of time, and then harvested for analysis of the myofibroblast markers and the key components of Wnt/β-catenin signaling. A small-molecule inhibitor, ICG-001, which specifically disrupts β-catenin signaling in a CBP-dependent fashion has been reported in previous literature^{26 (link)}. Both human and mouse LR-MSCs were pretreated with ICG-001 (Selleckchem, Houston, TX) for 1 h, followed by incubation with 10 ng/ml hTGF-β1 or TGF-β1 (PeproTech, Rocky Hill, NJ) for 24 h. The cells were collected for Q-PCR, Western blot analysis, and immunofluorescence staining.

The lentiviral expression plasmids pCDH-Smad2 and the SUMO site mutant pCDH-Smad2 were constructed by standard PCR-based strategies with the indicated primers (Table S1). Overexpressed cell lines were generated using a lentiviral system (System Biosciences). The virus was generated in HEK293TN cells by transfecting the packaging (psPAX2) and envelope (pMD2.G) plasmids. Cell culture media were collected 48 h after transfection and immediately transferred to target cells. The transduced cells were selected with puromycin for 48 h. Then 10 ng/ml hTGF-β1 (Cell Signaling) was added to HAECs and HUVECs that stably expressed Smad2w and SUMO mutant Smad2 for 3 days. Real-time PCR or immunoblotting was manipulated to detect the efficacy of overexpression.

Cells were seeded on the SISmuc as described above. After 3 days in culture, medium containing 2 ng/mL hTGF-β1 with a carrier (Cell Signaling, Danvers, MA, USA) was added to the models and renewed every 2 to 3 days for the remaining 11 days in culture.