Figure 2. Insulin impedes osteogenic differentiation of H-BMSCs by inhibiting autophagy and inducing senescence. H-BMSCs were incubated under normoglycemic or hyperglycemic conditions with or without of insulin. The amount of calcium accumulation was measured by Alizarin Red S staining after osteogenic inducement for 14 days (A). Quantitative analysis of calcium-bound staining was determined by comparison with calcium standards (B). H-BMSCs were cultured in normal or high-glucose medium. Rapamycin (RA) as positive control. Fluorescence detection of autophagosomes in H-BMSCs were transfected with the GFP-LC3 plasmid and cultured in serum deprivation conditions for 6 h (C). H-BMSCs cultured in normal or high-glucose medium with or without insulin. Western blot analysis showed the conversion of LC3-I into LC3-II after serum deprivation for 6 h (D). Protein bands were quantified and analyzed by densitometric analysis (E). H-BMSCs cultured in normal or high glucose medium with or without insulin for 3 days. H-BMSC senescence was measured by SA-β-Gal staining (F). The number of positive cells was calculated (G). NG, normoglycemic condition, HG, hyperglycemic condition. Data are presented as the mean ± standard deviation, n=3. *p<0.05, #p>0.05. Scale bar = 100 μm.