Recombinant human tgf β1

All animal care and laboratory procedures of this study were approved by the Ethical Committee of Harbin Medical University and all animal were obtained from the Experimental Animal Center of Second Affiliated Hospital of Harbin Medical University, China. Neonatal mouse (1–3 days old) hearts were prepared for finely minced and placed together in 0.25% trypsin to get single cell suspension. After the cells were filtered and centrifuged (1000 rpm/min, 5 min) and then resuspended in DMEM (HyClone, USA) containing 10% fetal bovine serum and 5% penicillin/streptomycin. Finally, the cells were plated into Petri dishes (60 mm) or other specifications of culture plate and cultured under a condition of 5% CO₂ and 95% air at 37 °C for 1.5 h. Then, cardiac fibroblasts preferential attached the bottom of petri dishes and new DMEM containing 10% fetal bovine serum and 5% penicillin/streptomycin was replaced in the Petri dishes to culture primary cardiac fibroblasts. The cardiac fibroblasts were pre-stimulated with choline (5 mM) for 1 h and then incubated with recombinant human TGF-β1 (20 ng/mL, Sigma-Aldrich Co., LLC, USA) for 48 h. Choline (1 mM) treatment was operate as describe previously [19 (link)]. The cells were used for the experiments.

Ang II, ET-1, PD123319, gallein (G_βγ inhibitor), and BQ788 were obtained from Tocris Bioscience (Ellisville, MO, USA). Recombinant human TGF-β1, valsartan, bosentan, ambrisentan, LY2109761, FR180204, and SB203580 were obtained from Sigma Aldrich (Saint Louis, MO, USA). SIS3 (Smad3 inhibitor) and FR900359 (G_αq inhibitor) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Fibroblast growth medium and related cell culture reagents were obtained from Promocell (Heidelberg, Germany).

One day before EMT induction, MCF-10A cells were placed in standard 6-well culture plates. Recombinant human TGF-β1 (Sigma-Aldrich) was added to the culture media at 5 ng/mL to induce EMT. Alternatively, the TGF-β receptor inhibitor SB-431542 (Sigma-Aldrich) was added to the culture media at 10 μM final concentration to enforce an epithelial phenotype on MCF-10A cells. Cell exposure to these conditions for 7 d ensured full effects^{46 (link)}. Cells re-plated for imaging or Western blot studies were cultured with no additives, 5 ng/mL TGF-β1, 10 μM SB-431532, or both agents together. We incubated cells with or without additives for 2 d before imaging or Western blot.

Cells were seeded on 6-well tissue culture plates at a density of 37,500 cells/cm². The cells were subjected to CCF or ICF stimulation in serum-free medium for 24 h. Subsequently, the culture medium was changed to osteogenic medium, which was normal growth medium supplemented with β-glycerophosphate (5 mM, cat. No. G9422, Sigma-Aldrich), l-ascorbic acid (50 μg/mL, cat. No. A-4034, Sigma-Aldrich), and dexamethasone (250 nM, cat. No. D8893, Sigma-Aldrich). The medium was changed every 48 h.
In other experiments, cells were seeded on 48-well tissue culture plates (cat. No. 3548, Costar^®, Corning) at a density of 37,500 cells/cm² and allowed to attach for 24 h. The cells were then starved in serum-free medium for 8 h and subsequently exposed to adenosine 5′-triphosphate disodium salt hydrate (ATP, cat. No. A6419, Sigma-Aldrich) or recombinant human TGF-β1 (cat. No. 616455, Calbiochem^®) for 24 h in serum-free culture medium. The cells were then maintained in osteogenic medium.