Figure 2
Influence of UGCG overexpression on mitochondrial ROS and mass. (A) Mitochondrial reactive oxygen species (ROS) levels were quantified using the fluorogenic reagent MitoSOX. Data are presented as a mean of n = 3 ± SEM. (B) Mitochondrial membrane potential was analyzed by flow cytometry utilizing 5,5,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazoyl-carbocyanine iodide (JC-1). Data are presented as a mean of n = 3 ± SEM. (C) Representative images of n = 3 of JC-1 staining by immunocytochemistry. From each of the biological samples at least 5 images were generated. (D) Mitochondrial mass determined by flow cytometry using nonyl acridine orange (NAO). Data are presented as a mean of n = 4 ± SEM. (E) Analysis of translocase of the outer mitochondrial membrane 20 (TOM20) protein concentration by Western blot analysis. Protein expression is related to the housekeeper Hsp90 and control cells. Data are presented as a mean of n = 6 ± SEM. (F) Densitometrical analysis of oxidative phosphorylation (OXPHOS) complexes I-V protein concentrations by Western blot analysis. Protein expression is related to Ponceau dye. Data are presented as a mean of n = 3 ± SEM. (G) Representative image of the OXPHOS protein Western Blot analysis (upper part) and Ponceau staining (lower part). (H) The mitochondrial DNA (mtDNA) copy number was determined using the NovaQUANT Human Mitochondrial to Nuclear DNA Ratio Kit. Data are related to MCF-7/empty and presented as a mean of n = 7 ± SEM. *p ≤ 0.05, **p ≤ 0.01.
