Fig. 1: LPS-induced increase in mitochondrial membrane potential drives mitochondrial superoxide production.
a,b, Mitochondrial superoxide measured with MitoNeoD in BMDMs stimulated with LPS for 3–24 h, treated with 10 μM MitoParaquat for 30 min or nonstimulated (NS). NS BMDMs were either left for 24 h before MitoNeoD treatment (NS at 24 h) or immediately treated with MitoNeoD (NS 0 h). Representative images from confocal live-cell imaging show MitoNeoOH, the oxidized product of MitoNeoD and MitoTracker Deep Red FM. Fluorescence intensity of MitoNeoOH, measured as absolute intensity (arbitrary units (a.u.)) (n = 3 (NS, 6 h, 9 h, MitoPQ, NS at 24 h), n = 6 (3 h, 18 h, 24 h)). c,d, Succinate and itaconate measured by mass spectrometry in BMDMs treated with LPS for 3–24 h or NS (n = 6). e–g, ∆ψm measured with TMRM normalized to MitoTracker Deep Red FM in BMDMs stimulated with LPS for 3–24 h or NS and/or treated with 5 μM oligomycin in the last 30 min of LPS treatment or for 30 min in NS cells from confocal live-cell imaging (n = 6 (3 h, 6 h, 18 h, 24 h), n = 9 (NS, 9 h)) (NS, P = 0.00006; 3 h P = 9 × 10−9; 18 h, P = 1 × 10−5; 24 h, P = 8 × 10−5). h,i, Mitochondrial superoxide production measured with MitoNeoD from confocal live-cell imaging in BMDMs stimulated with LPS for 24 h and/or treated with 5 μM oligomycin in the last 30 min of LPS treatment or for 30 min in NS cells. Fluorescence intensity of MitoNeoOH is measured as absolute intensity (a.u.) (n = 6). Scale bars on all representative images, 20 μm. j, The ATP/ADP ratio measured per million BMDMs stimulated with LPS for 3–24 h or NS and/or treated with 5 μM oligomycin in the last 30 min of LPS treatment or for 30 min in NS cells (n = 3) (NS, 0.5 h, 1 h, 2 h and 3 h, P < 1 × 10−15). All data are mean ± s.e.m. of biological replicates. P values displayed above graphs were calculated using two-tailed Student’s t-test for paired comparisons or one-way or two-way analysis of variance (ANOVA) for multiple comparisons.
