The BMMSCs were divided into 4 groups as follows: (1) control group: the BMMSCs maintained only in basal media were used as the assay control; (2) TGF-β1 group: the BMMSCs were exposed to 10 ng/mL TGF-β1 (PeproTech, NJ, USA) for 14 days [12 (link)]; (3) 5-AZA group: the BMMSCs were exposed to 10 μmol/L of 5-AZA (Sigma-Aldrich Co. LLC, MO, USA) for 24 h and maintained in basal media alone for the next 28 days [14 (link)]; (4) combined low dose treatment group of TGF-β1 and 5-AZA: the BMMSCs were treated with 5 μmol/L 5-AZA and 5 ng/mL TGF-β1 for 24 h, then washed twice with PBS, and maintained with 5 ng/mL TGF-β1 alone for up to 14 days. For analysis of the mechanisms involved in the differentiation, a specific inhibitor of ErK, U0126 (10 μmol/L, Promega Corporation, Beijing, China), was added to the medium until the cells were collected.
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