Extended Data Fig. 1: ROS generation by RET from site I_Q is increased in the liver but not the skeletal muscle of obese mice.

(A) Liver sections from wildtype (wt) and ob/ob mice stained with H&E. Obese hepatocytes contain several lipid vacuoles (arrow) and small foci of inflammation (asterisk). Scale bar, 200 µm. (B) Immunoblot analysis and (C) quantification of 4-HNE as an oxidative stress marker in liver homogenates from wt and ob/ob mice. n = 6 livers per group (***p = 0.001, unpaired t-test). Area used for quantification is shown in Supplementary Fig. 1. (D) Immunoblot analysis and (E) quantification of peroxiredoxin 3 (PRDX3) in liver homogenates from wt and ob/ob mice. n = 4 per group (**p = 0.002, unpaired t-test). (F) Quantitative proteomics of PRDX3 in liver isolated mitochondria from wt and ob/ob mice. n = 9 mito isolations from n = 9 mice per group (*p = 0.011, unpaired t-test). (G) Schematic to illustrate the substrates and inhibitors used to assess the two modes of mROS generation from complex I: forward electron transport from site I_F (FET, left) and reverse electron transport (RET, right) from site I_Q. (H) Representative Amplex UltraRed traces showing that rotenone blocks mROS during succinate oxidation. The rate difference between minus and plus rotenone defines RET, which is higher in mitochondria isolated from ob/ob livers. Representative of n = 13 independent experiments. (I) Representative Amplex UltraRed traces and (J) quantification showing that 5 µM S1QEL 2.2, 2 µM rotenone, 2 µM piericidin A, and 1 µM FCCP decrease the rate of mROS production during RET induced by succinate oxidation in the presence of oligomycin in isolated mitochondria. n = 5 independent experiments, except piericidin A (n = 4) and S1QEL (n = 3) (****p < 0.0001, two-way ANOVA, Dunnett’s post hoc test) (K) Relative mROS production by RET from wt and ob/ob liver mitochondria using the compounds in (J). n = 3 mito isolations from n = 3 mice per group (*p < 0.05, multiple paired t-test not adjusted for multiple comparisons). (L) S1QEL 2.2 (0.15−10 µM) does not inhibit Hepa 1-6 oxygen consumption rates (OCR). n = 5 independent experiments, except DMSO (n = 8) and S1QEL 0.15 and 0.3 µM (n = 4). ns, p = 0.3268 two-way ANOVA, Dunnett’s post hoc test). (M) Effect of S1QEL 2.2 (0.01−10 µM) on mROS production during RET in liver-isolated mitochondria from wildtype and ob/ob mice. n = 3 mito isolations from n = 3 mice per group (*p < 0.0001, two-way ANOVA). (N) Maximum capacity of superoxide/H₂O₂ production from skeletal muscle-isolated mitochondria from lean wt (n = 12) and ob/ob mice (n = 12). Pooled from four independent experiments (*p = 0.019, multiple unpaired t-test not adjusted for multiple comparisons). Data are individual values and means ± SEM. All t-tests were two-tailed.

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