Fig. 4
AGEs affect the osteogenic differentiation and m6A modification level of BMSCs. A ELISA quantification of serum AGEs level of control and DM mice with or without experimental periodontitis (n = 6). B Proliferation of BMSCs stimulated with different doses of AGEs (0, 50, 100 or 150 µg/ml) was determined by CCK-8 assay (n = 3). C Detection of apoptotic BMSCs through DAPI and TUNEL staining. The percentage of TUNEL-positive BMSCs was counted from three random microscopic fields (n = 3). Scale bar = 200 μm. D Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) analysis of the expression of Runx2, Col1a, ALP, Bglap, SOST and Dkk1 in BMSCs untreated or treated with AGEs under osteogenic induction for 3 days (n = 3). E Representative images of alkaline phosphatase (ALP) staining and quantification of ALP activity in BMSCs at day 7 after exposure to osteogenic induction with or without AGEs (n = 3). Scale bars = 1000 μm. F Representative images of Alizarin red S (ARS) staining and quantification of mineralization nodules in BMSCs at day 21 after exposure to osteogenic induction with or without AGEs (n = 3). Scale bars = 1000 μm. G The m6A methylation levels in BMSCs at day 10 after exposure to osteogenic induction with or without AGEs were detected by m6A dot blot assays. Input RNA was assessed by methylene blue staining (left panel), and the level of m6A modification was determined by the intensity of dot immunoblotting (right panel). H Western blot (WB) analysis of the protein level of Runx2, Bglap, SOST and FTO in BMSCs untreated or treated with AGEs under osteogenic induction for 5 days (n = 3). I RT-qPCR analysis of the expression of Mettl3, Mettl14, FTO, Alkbh5, Ythdf1 and Ythdf2 in BMSCs untreated or treated with AGEs under osteogenic induction for 3 days (n = 3). Data are expressed as the mean ± SEM. ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001