Fig. 6

Autophagy inhibition by 3-MA prevented 2 nM RAPA-induced autophagy enhanced osteogenesis. A Western blot of LC3, BECN1, and p62 in BMSCs with or without 3-MA treatment for 24 h. B Quantitative BECN1and p62 protein levels and the LC3-II/LC3-I ratio. C mRNA expression levels of Becn1, Lc3, and Atg5. D Protein expression of ALP, BSP, and RUNX2 on days 7 and 14 after osteogenic differentiation. Quantitative protein expression of ALP, BSP, and RUNX2 on days 7 (E) and 14 (F). G Representative ALP staining images of two groups at two time points (scale bar = 200 μm) and ARS staining images on day 14 (scale bar = 100 μm). H Quantitative analysis of ARS staining of BMSCs. The mRNA expression levels of Alpl, Ibsp, Runx2, Spp1, and Col1a1 on days 7 (I) and 14 (J) after osteogenic differentiation. *p < 0.05, **p < 0.01, ***p < 0.001 (vs. control or 2 nM RAPA)