Figure 4.

SOD2 is required for osteogenic differentiation. (a) MC3T3-E1 cells were induced to differentiate for 48 h with or without 10 μM FCCP, and mRNA levels of Runx2 and BSP were tested. (b) Cells were induced to differentiate for 14 days with or without 10 μM FCCP, and ARS and ALP staining was performed. (c) SOD2 protein content from cells that stably expressed SOD2 shRNA was tested by western blot. (d) Mitochondrial superoxide levels were visualized by fluorescence staining. (e) Mitochondrial biogenesis regulators and mitochondrial complex subunits were analyzed by western blot. (f) ARS and ALP staining of SOD2-knockdown cells differentiated for 7 days. (g) mRNA levels of Runx2, Osterix, ALP, and BSP from SOD2-knockdown cells differentiated for 2 days. Data are presented as the mean±S.E.M. from at least three independent experiments. *P<0.05, **P<0.01 versus relative control