Figure 4

Upregulation of Lon increases mitochondrial ROS through the stabilization and upregulation of NDUFS8 of complex I by Lon. (a) Mitochondrial superoxide generation in 293/Lon cells was inhibited by rotenone (2 μM, 3 h). MitoSox staining and fluorescence intensity of the cells was measured. (b) Upregulation of Lon increases the protein level of NDUFS3 and NDUFS8. 293T, OEC-M1, or FADU cells were stably transfected with plasmid encoding Lon. The cell extracts were immunoblotted with indicated antibodies and antibody to actin as a loading control. (c) Expression level of Lon and NDUFS8 in 293 cells overexpressing Lon and NDUFS8-shRNA. 293/Lon cells were retrovirally infected with pMKO vector or pMKO expressing shRNAs for human NDUFS8. Immunoblots were probed with indicated antibodies and anti-actin antibody acts as a loading control. The expression of NDUFS8 with or without infection of shRNAs was shown. (d) Mitochondrial superoxide generation in 293 cells overexpressing Lon was inhibited by NDUFS8-shRNA. MitoSox staining and fluorescence intensity of the cells was measured. (e) Lon interacts with NDUFS8 shown by Co-IP. OEC-M1 or FADU cells were stably transfected with plasmid encoding Myc-Lon followed by Co-IP with anti-Lon and anti-NDUFS8, respectively. The immunoprecipitation complex was analyzed by western blotting using indicated antibodies. (f) The complex formation between mitochondrial Lon and NDUFS8 was verified by confocal immunofluorescence OEC-M1/Lon cells were fixed and immunostained by anti-NDUFS8 (green) and anti-Lon (red) antibodies. DNA was stained with DAPI (blue). (g) Downregulation of Lon decreases the protein level of NDUFS8. 293 and OEC-M1 cells were retrovirally infected with pMKO vector or pMKO expressing shRNAs for GFP or Lon. Immunoblots were probed with indicated antibodies and anti-actin antibody acts as a loading control. The expression of NDUFS8 with or without infection of Lon-shRNA was shown. (h) The proteolytic activity of Lon is not a major factor to the stabilization of NDUFS8 by Lon. 293T cells were transfected with wild-type pcDNA3-Lon (WT), a proteolytic mutant (LonS855 A) or pcDNA3 vector and treated with H₂O₂ (200 μM for 4 h) or not. Exogenous overexpression of Lon was verified by western blotting with anti-Lon antibody. Western blotting was performed by using indicated antibodies. An antibody to actin was used as a loading control. The number below Aconitase and TFAM blot represents the band intensity normalized against actin. The numbers shown in the bottom represent Lane 1 to 6