Fig. 3

nZnO induces autophagy during osteogenic differentiation of MSCs. (A) Volcano map of the RNA-seq data of MSCs in the control versus nZnO (5 µg/mL) groups after osteogenic differentiation for 72 h calculated by DESeq2 (n = 3 in each). (B) GOCircle plot of GO enrichment analysis of the DEGs. (C) KEGG enrichment analysis of DEGs. (D) Transmission electron microscopy of MSCs under control and nZnO treatment for 72 h. Arrows indicate autophagosomes; Scale bars: 1 μm, Magnified Scale bars: 500 nm. (E) Autophagic flux detection for MSCs transfected with Ad-mCherry-GFP-LC3B under control and nZnO treatment; Scale bars: 40 μm. (F) Western blot analysis of autophagic activation in MSCs under different concentrations of nZnO treatment at osteogenic differentiation for 72 h. Full-length blots were presented in supplementary materials 2: Fig.S6. (G) ALP staining and ARS staining of MSCs after osteogenic differentiation for 7 days and 14 days treated with autophagy inhibitors chloroquine (CQ, 10 µM) under control and nZnO (5 µg/mL) conditions, respectively. Scale bars: 2 mm, Magnified Scale bars: 200 μm. (H) ALP activity assay of MSCs cells after osteogenic differentiation for 7 days treated with autophagy inhibitors CQ under control and nZnO (5 µg/mL) conditions (n = 6), and quantitative analysis of the Alizarin Red staining in G (n = 6). (I) Western blot analysis of autophagic activation in MSCs cells after osteogenic differentiation for 72 h treated with autophagy inhibitors CQ under control and nZnO (5 µg/mL) conditions. Full-length blots were presented in supplementary materials 2: Fig.S7. (J) Autophagic flux detection for MSCs transfected with Ad-mCherry-GFP-LC3B treated with autophagy inhibitors CQ under control and nZnO (5 µg/mL) conditions; Scale bars: 40 μm. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 relative to control