Fig. 3

Effect of Pink1 siRNA on mitochondrial homeostasis and ROS production during osteoblast differentiation. a Expression levels of mitochondria functional unit (Tom20), profusion (Mfn1), pro-fission (Drp1 and Fis1), and autophagy (LC3ii and p62) related proteins in MC3T3-E1 cells after osteogenic induction. b The effects of Pink1 siRNA on protein levels of mitochondria functional unit, profusion, pro-fission, and autophagy-related proteins in cells after differentiation for 3 days. c mtDNA copy number in cells after treatment with Pink1 siRNA. d The effects of Pink1 siRNA on Mitochondrial respiration, reflected by the oxygen consumption rate (OCR) level in cells after differentiation for 3 days. The oxygen consumption rate (OCR) was analyzed using a Seahorse XF-24 analyzer. Rates of basal respiration were quantified by normalization of OCR levels to total protein levels obtained from O.D. values. e Mitochondrial membrane potential (ΔΨm) was studied by measuring JC-1 uptake in MC3T3-E1 cells after osteogenic induction with or without Pink1 siRNA treatment. f Representative confocal images of MitoTracker™ (red) in MC3T3-E1 cells with or without treatment with Pink1 siRNA. Scale bar = 20 μm. g Representative confocal images of mtKeima in MC3T3-E1 cells with or without treatment with Pink1 siRNA. Scale bar = 20 μm. h–i) Representative confocal images of 2,7-dichlorodihydrofluorescein diacetate (H2-DCFDA; green) and MitoSOX™ (red), showing mitochondrial and intracellular ROS production in MC3T3-E1 cells with or without treatment with Pink1 siRNA. mtDNA, mitochondrial DNA; CCCP, carbonyl cyanide m-chlorophenyl hydrazone. Data have been expressed as mean ± SEM; *P < 0.05; **P < 0.01; n = 4; scale bar = 50 μm