Fig. 4

AMPK-ULK1 signaling pathway participates in nZnO induced autophagy and osteogenic differentiation. (A) GSEA analysis demonstrated significant enrichment of AMPK-regulated genes in the nZnO (5 µg/mL) versus control group after osteogenic differentiation for 72 h. (B) Western blot analysis of AMPK and ULK1 activation in MSCs under different concentrations of nZnO for after osteogenic differentiation for 72 h. Full-length blots were presented in supplementary materials 2: Fig.S8. (C) Relative protein expression determined in B (n = 3). (D) Western blot analysis of AMPK-ULK1 and autophagic activation in MSCs after osteogenic differentiation for 72 h treated with AMPK inhibitor compound C (C.C, 1 µM) or agonist AICAR (Aic, 10 µM) under control and nZnO (5 µg/mL) conditions. Full-length blots were presented in supplementary materials 2: Fig.S9. (E) ALP staining and ARS staining of MSCs after osteogenic differentiation for 7 days and 14 days treated with AMPK inhibitor C.C or agonist Aic under control and nZnO (5 µg/mL) conditions, respectively. Scale bars: 2 mm, Magnified Scale bars: 200 μm. (F) ALP activity assay of MSCs after osteogenic differentiation for 7 days treated with AMPK inhibitor C.C or agonist Aic under control and nZnO (5 µg/mL) conditions (n = 3), and quantitative analysis of the Alizarin Red staining in D (n = 6). (G) Western blot analysis of AMPK-ULK1 and autophagic activation in MSCs after osteogenic differentiation for 72 h treated with ULK1 inhibitor SBI-0206965 (SBI, 1 µM) or agonist BL-918 (BL, 10 µM) under control and nZnO (5 µg/mL) conditions. Full-length blots were presented in supplementary materials 2: Fig.S10. (H) ALP staining and ARS staining of MSCs after osteogenic differentiation for 7 days and 14 days treated with ULK1 inhibitor SBI or agonist BL under control and nZnO (5 µg/mL) conditions, respectively. Scale bars: 2 mm, Magnified Scale bars: 200 μm. (I) ALP activity assay of MSCs after osteogenic differentiation for 7 days treated with ULK1 inhibitor SBI or agonist BL under control and nZnO (5 µg/mL) conditions (n = 3), and quantitative analysis of the Alizarin Red staining in G (n = 6). Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control