Fig. 2: Reduction of the CoQ pool maintained by succinate oxidation is required for mitochondrial superoxide production.
a,b, The redox state of the CoQ pool was measured by mass spectrometry in WT and AOX-expressing BMDMs that were NS, stimulated with LPS for 24 h, treated with 5 μM antimycin A for 15 min or stimulated with LPS and 100 μM n-PG for 24 h (n = 9 (24 h LPS), n = 12 (NS, antimycin A)) (NS versus antimycin A, P = 8 × 10−12; LPS versus antimycin A, P = 6 × 10-12; WT LPS versus WT AA, P = 5 × 10-6; WT AA versus AOX NS, P = 7 × 10-8). c,d, Mitochondrial superoxide production measured in NS WT BMDMs, WT BMDMs stimulated with LPS for 24 h and in AOX-expressing BMDMs that were NS, stimulated with LPS for 24 h with or without 100 μM n-PG or treated with 10 μM MitoPQ for 30 min from confocal live-cell imaging. MitoNeoOH fluorescence is measured as absolute intensity with arbitrary units (a.u.) (n = 3 (WT NS, AOX LPS + n-PG, AOX + MitoPQ) n = 6 (WT LPS, AOX NS, AOX LPS)) (WT LPS versus AOX NS P = 3 × 10−5; WT LPS versus AOX LPS P = 3 × 10−5). e,f, ∆ψm measured using TMRM normalized to MitoTracker Deep Red FM in WT and AOX-expressing BMDMs that were NS or stimulated with LPS for 24 h from confocal live-cell imaging (n = 3). g–j, NS BMDMs or BMDMs stimulated with LPS for 24 h and BMDMs stimulated with LPS for 12 h and then for a further 12 h with the addition of 10 mM DMM, 200 μM TTFA or 0.5 μM AA5. Succinate measured by mass spectrometry (n = 3) (NS versus TTFA, P = 3 × 10−6; NS versus AA5, P = 2 × 10−7) (g). The redox state of the CoQ pool measured by mass spectrometry (n = 3) (h). i,j, Mitochondrial superoxide production measured with MitoNeoD from confocal live-cell imaging. MitoNeoOH fluorescence is measured as absolute intensity (a.u.) (n = 3). Scale bars on all representative images, 20 μm. All data are mean ± s.e.m. of biological replicates. P values displayed above graphs calculated using one-way or two-way ANOVA.
