Figure 4. MDA formation and content of 4-HNE adducts in tissues from mtDNA mutator mice non-treated and treated with SkQ1. (A) MDA formation in kidney lysate exposed to normal atmospheric oxygen concentration and pressure at 37 °C. (B) Levels of MDA formed in liver, brain and kidney lysates after 40 min of exposure of tissue to normal atmospheric oxygen concentration and pressure. (C) Representative immunoblot analysis of 4-hydroxynonenal (4-HNE) adducts in kidney (left panel), liver (middle panel) and gastrocnemius skeletal muscle (right panel) tissue lysate from non-treated (M) and SkQ1-treated (S) mtDNA mutator mice (15 µg protein/lane). Numbers 1, 2, 3 indicate age-and gender-matched samples. Validation of the assay is presented in Fig. S1A. Loading control was performed by Ponceau Red staining (shown for kidney in Fig. S1C). (D) Profile of 4-HNE-adducts in kidney lysate. (E) Relative 4-HNE-adducts in kidney, liver, skeletal muscle (SKM) and brain tissue lysates. Tissue samples were collected in parallel from non-treated and SkQ1-treated mice (268–300 days old, both genders) in the paired death experimental setup. In B and E, for each pair, the mean level of 4-HNE-adducts in the non-treated mouse was set to 100% (indicated as dashed line) and the amount in the paired (age- and gender-matched) SkQ1-treated mouse was expressed relative to this. The means ± S.E. for 4-7 mice are shown. * and ** in A, B and E indicate statistical significance between SkQ1-treated and non-treated mice (p < 0.05 and p < 0.01, respectively).