UCP2 deficiency does not affect proton leak kinetics or ROS production.

<p>(A) Flow-force relationship in heart mitochondria of 25-week-old wild type (wt), mtDNA mutator (mut), UCP2-deficient wild type (ko) and mtDNA mutator (dm) mice examined in the presence of succinate and increasing amounts of malonate (n = 6). Data points indicate mean levels ± standard error of the mean (S.E.M.). (B) Hydrogen peroxide production rate in heart mitochondria in the presence of succinate (S); Pyruvate-Glutamate-Malate (PGM) or Pyruvate-Glutamate-Malate-Succinate (PGMS) as substrates. Measurement in the presence of succinate alone detects reverse flow ROS production from both Complex I and Complex III. When mitochondria are incubated in the presence of pyruvate-glutamate-malate (PGM), the ROS production mainly originates from complex III and therefore it is usually lower. Treatment with Antimycin A (+AA), an inhibitor of CO III, was used as a positive control, to further induce ROS production (n = 4–5). Bars indicate mean level ± standard error of the mean (S.E.M.). (C) Western blot analyses of Manganese Superoxide Dismutase (MnSOD) in heart lysates of 25- and 40-week-old mice (n = 4). (D) Analysis of 4-Hydroxynonenal as measure for oxidative stress induced lipid peroxidation in isolated mitochondria (upper panel) and tissue lysates (lower panel) of 25-week-old mouse hearts. The mitochondrial Complex II 70 kDa protein (C II) was used as loading control in isolated mitochondria and the Heat shock 70 kDa protein 8 (HSC 70) in heart tissue lysates (n = 4). (E) Fold change of <i>Ucp2</i> transcript levels in primary MEFs (P1–P3). (F) ROS production in intact cells was assessed by flow cytometric analyses of primary (passage 1–3) mouse embryonic fibroblasts (MEFs) stained with CM-H₂DCFDA that upon oxidation by ROS, particularly hydrogen peroxide (H₂O₂) and the hydroxyl radical (·OH), yields the fluorescent DCF product. Data are expressed as median values of fluorescence intensity ± standard error of the mean (S.E.M.). Asterisks indicate level of statistical significance (*p<0.05 **p<0.01 ***p<0.001, Student's <i>t</i>-test).