Recombinant human tgf β1 rhtgf β1

5(S), 6(R)-Lipoxin A4 (LXA4) and 5(S), 6(R)-7-trihydroxymethyl Heptanoate (BML-111) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against E-cadherin, N-cadherin, Vimentin, and Snail were obtained from Cell Signaling Technology (Boston, MA, USA). The antibodies against FPRL1 and 15-LOX were purchased from Abcam (Cambridge, UK). The anti-TGF-β1 neutralizing antibody was obtained from R&D Systems (Minneapolis, MN, USA), and recombinant human TGF-β1 (rhTGF-β1) was obtained from PeproTech (Rocky Hill, NJ, USA). Anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

To silence TGF‐β1, the cells were transfected with a TGF‐β1 shRNA or with a scramble control (GenePharma, Hi‐Tech Park, Shanghai, China) using a Lipofectamine reagent (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions. A stable TGF‐β1‐silenced cell line was obtained using puromycin selection. MCF‐7 cells were treated with 10 ng·mL⁻¹ of recombinant human TGF‐β1 (rhTGF‐β1) (PeproTech, Rocky Hill, NJ, USA) for 24 h and then removed by replacing cultural medium.

Bone tissue was disintegrated mechanically into millimeter-sized pieces. After washing 3 to 4 times with phosphate buffered saline (PBS), the pieces were incubated in a 0.7% collagenase II solution (Biochrom, Berlin, Germany) for about 1 h at 37 °C. Cancellous bone pieces were washed with PBS, and released phOBs were transferred to cell culture flasks in the culture medium (MEM/Ham’s F12, 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μM l-ascorbate-2-phosphate, 50 μM β-glycerol phosphate) for expansion. The medium was changed every 3–4 days. For the experiments, the cells (passage 3) were seeded at a density of 20,000 cells/cm² in the culture medium. After 3 days, the culture medium was replaced by osteogenic differentiation medium (MEM/Ham’s F12, 1% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, 200 μM l-ascorbate-2-phosphate, 5 mM β-glycerol phosphate, 25 mM HEPES, 1.5 mM CaCl₂, 100 nM dexamethasone) ± 5 ng/mL recombinant human TGF-β₁ (rhTGF-β₁) (Peprotech, Hamburg, Germany) [13 (link)].