Research Paper Volume 12, Issue 3 pp 2084—2100

Insulin impedes osteogenesis of BMSCs by inhibiting autophagy and promoting premature senescence via the TGF-β1 pathway

Figure 3. TGF-β1 participates in insulin inhibiting autophagy and osteogenic differentiation of H-BMSCs. H-BMSCs were cultured in normal or high-glucose medium with or without insulin. The expression of TGF-β1 in H-BMSCs was detected by western blot (A). Protein bands were quantified and analyzed (B). H-BMSCs were incubated in hyperglycemic and insulin conditions with or without hrTGF-β1. Rapamycin (RA) as positive control. The protein level expression of LC3 and P62 were detected by western blot (C). Protein bands were quantified and analyzed by densitometric analysis (D, E). H-BMSCs were cultured in osteogenic medium for 7 days with or without insulin under normoglycemic or hyperglycemic conditions stimulated with SB431542 or hrTGF-β1. mRNA level expression of Alp (F), Runx2 (G), Ocn (H), and Opn (I) was detected by real-time PCR. NG, normoglycemic condition, HG, hyperglycemic condition. Data are presented as the mean ± standard deviation, n=3. *p<0.05, $p<0.05, #p>0.05, * in F-I as compared to insulin-untreated cells, $ and # in F-I as compared to corresponding insulin-treated cell.