Fig. 3: LPS-induced mitochondrial superoxide is produced by reverse electron transport at complex I.
a–h, Mitochondrial superoxide production measured with MitoNeoD from confocal live-cell imaging. Fluorescence intensity of MitoNeoOH is measured as absolute intensity with arbitrary units (a.u.). BMDMs stimulated with LPS for 24 h were treated with 0.5 μM rotenone in the last 30 min of LPS treatment or for 30 min with 0.5 μM rotenone or 5 μM oligomycin in NS cells (n = 3) (NS versus LPS, P = 7 × 10−6; LPS versus LPS + rotenone, P = 2 × 10−5) (a–d). BMDMs stimulated with LPS for 24 h were treated with 5 μM antimycin A in the last 30 min of LPS treatment or for 30 min in NS cells (n = 3) (e,f). BMDMs from WT and ND6 P25L mice stimulated with LPS for 24 h, NS or treated with 5 μM MitoPQ for 30 min (n = 3 (ND6P25L MitoPQ), n = 6 (WT NS, WT LPS, ND6P25L NS, ND6P25L LPS)) (g,h). i,j, ∆ψm measured using TMRM normalized to MitoTracker Deep Red FM in BMDMs from WT and ND6P25L mice that were treated with 5 μM oligomycin for 30 min or stimulated with LPS for 24 h from confocal live-cell imaging (n = 3). Scale bars on all representative images, 20 μm. k, The redox state of the CoQ pool measured by mass spectrometry in BMDMs from WT and ND6P25L mice that were NS, stimulated with LPS for 24 h or treated with 5 μM antimycin A for 15 min (n = 3). All data are mean ± s.e.m. of biological replicates. P values displayed above graphs calculated using two-tailed Student’s t-test for paired comparisons or one-way or two-way ANOVA for multiple comparisons.
