Fig. 3

The diabetic microenvironment induces mitophagy impairment in osteoblasts. a, b Enrichment Analysis for differentially expressed genes between the CTRL and HFD&STZ groups using GO and KEGG databases. c Detection of osteoblast mitochondrial ultrastructure by transmission electron microscopy (TEM) in femurs from CTRL and HFD&STZ mice. d Visualization of mitochondria in MC3T3-E1 cells after HGPA treatment using Mitotracker Green dye. e Representative images of JC-1 staining in CTRL and HGPA-treated MC3T3-E1 cells. Red fluorescence represented intact mitochondria with normal membrane potential and green fluorescence represented impaired mitochondria with reduced membrane potential. Scale bar, 200 μm. f Representative images of MitoTracker-labeled mitochondria (deep red), Mtphagy-labeled mitophagy process (red), and Lyso Dye-labeled lysosomes (green), of which the co-localization indicates the occurrence of mitophagy. Scale bar, 50 μm. g, h Western blot images and quantification of LC3 in MC3T3-E1 cells treated with gradient concentrations of HGPA, with or without Baf-A1 (10 nmol/L). i Representative IF staining of TOM20 (red), PRKN (green), and MitoTracker (deep red) in MC3T3-E1 cells from CTRL and HGPA groups stimulated by CCCP for 6 h. Scale bar, 50 μm. j Western blot results of mitophagy-related proteins including PINK1, PRKN, SQSTM1, and LC3 in MC3T3-E1 cells stimulated by HGPA for various duration. k Western blot analysis of PINK1 and PRKN in bone from CTRL and HFD&STZ mice. l qPCR analysis of Pink1 and Prkn genes. n = 6 per group. Data presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001