Tgf β1

Human GFs were cultured in DMEM (Lonza) supplemented with 10% FBS (Lonza) and 0.1% gentamicin sulfate (10 mg/ml; Euroclone) at 37°C in 5% CO₂ atmosphere. Cells between the fourth and tenth passages were used for the following experiments. At Passage 6 cells were seeded overnight with medium containing 1% of FBS (Lonza) and then were treated with or without TGF-β1 for 24, 48, and 72 h. Medium was removed 24 h after seeding and fibroblasts were incubated for 24, 48, and 72 h with DMEM plus with 1% FBS (Lonza) serum alone or supplemented with 10 ng/ml TGF-β1 (S.I.A.L.). Myofibroblasts phenotype was confirmed by flow cytometry, confocal microscopy, and Western blot for α-SMA, Vimentin, E-cadherin, β-catenin, and Smad 2/3. Once the obtained cell differentiation cells were collected to perform all experiments, the untreated cells were used as negative controls (CTRL).

MC3T3.E1 cells/3T3 F442A cells were seeded in 6-well plates until 70–80% confluence and then cells were induced for osteogenic or adipogenic differentiation, as described above. After cells were transfected with the miR-6315 agomir or negative control for 48 h, as described above, the differentiation medium was supplemented with/without 1 ng/mL of TGF-β1 (Lonza, Brooklyn, VIC, Australia). Treatment effects on expression of the TGF-β/Smad2 signalling pathway target genes were analysed by RT-qPCR and Western blot, as described below.