Extended Data Fig. 6: Ectopic expression of alternative oxidase in BMDMs.
a, Transgenic expression of Ciona intestinalis alternative oxidase (AOX) in the mitochondrial inner membrane where it uses electrons from the CoQ pool to reduce oxygen to water without pumping protons or generating ROS. N-propyl gallate (n-PG) inhibits AOX. b,c, Succinate, itaconate and extracellular lactate measured by mass spectrometry in wild-type (WT) BMDMs and BMDMs expressing AOX that were nonstimulated (NS) or stimulated with LPS for 3 h, 6 h or 24 h (n = 3). d, Nitrite levels in WT BMDMs and BMDMs expressing AOX that were NS or stimulated with LPS for 3 h, 6 h or 24 h (n = 3). e,f, Malonate and succinate in BMDMs that were NS or stimulated with LPS for 12 h and then for a further 12 h with the addition of 10 mM dimethyl malonate (DMM), 200 μM thenoyltrifluoroacetone (TTFA) or 0.5 μM atpenin A5 (AA5) (n = 3). P values for malonate (NS DMM vs Control, P = 3×10-6; vs TTFA, P = 4×10-6; vs AA5, P = 1×10-5; LPS DMM vs Control, P = 1x-11; vs TTFA, P = 8×10-12; vs AA5, P = 8×10-12). P values for succinate (NS Control vs TTFA, P = 5×10-7; vs AA5, 4×10-10). g, ∆ψm measured by TMRM normalized to MitoTracker Deep Red FM with Arbitrary Units (A.U.) from confocal live cell imaging in 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 (n = 3). Scale bars are 20 μm for all confocal images. h, THP-1 cells were differentiated to macrophage-like cells and treated with either 0.1 μM rotenone or 0.1 μM AA5 in the last 15 min of LPS treatment (n = 3 (NS, LPS + Rotenone, LPS + AA5), n = 6 (24 h LPS)). Mitochondrial superoxide production measured with MitoNeoD from confocal live cell imaging. Fluorescence intensity of MitoNeoOH is measured as absolute intensity (A.U.). Scale bars on all representative images are 20 μm. 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 ANOVA for multiple comparisons.
