Fig. 2: The TCA Break is due to NO targeting of mitochondrial aconitase.
a WT and Nos2−/− BMDMs were stimulated for 8 h and mRNA was extracted from total cell lysates and analyzed by qPCR for Idh1 (n = 6) (two-way ANOVA, Sidak’s post-tests; p-values = 0.033, 0.0093). b Whole-cell lysates of macrophages derived from two independent WT and Nos2−/− mice were analyzed by western blot for cytosolic and mitochondrial aconitase (ACO 1-2). β-actin was used as loading control. c, d Aconitase 2 and total aconitase (aconitase 1) enzymatic activities in WT vs. Nos2−/− BMDMs stimulated over night (O.N.). Where indicated cells were treated with NOS2 inhibitor Aminoguanidine (AG) 1 h prior to stimulation (n = 6). e Damaging effect of endogenously produced or exogenously provided NO (DETA/NO) on activity of aconitase 2 (n = 3). f Representative Seahorse analysis of oxygen consumption rates (OCR) in permeabilized WT and AG-pretreated BMDMs stimulated O.N. Citrate, tartronate, ADP and PMP were co-injected (first event marker). Isocitrate was injected at the second event marker and rotenone (rot) was injected lastly. g Quantifications (percentage relative to ctrl cells) of exogenous citrate and isocitrate-dependent state 3 OCR in WT vs. Nos2−/− (n = 6). Data were analyzed by one-way (c–e) or two-way ANOVA (f, g), (p < 0.0001) with Tukey’s post-tests. h Nos2−/− BMDMs were stimulated with LPS + IFNγ in the presence of either DETA/NO (500 μM) or FA. Metabolites were quantified by ESI-LC/MS-MS and are reported as normalized total ng or peak area. Data (n = 6) were analyzed by one-way ANOVA with Dunnett’s post-tests. i Bar graphs showing quantified, protein normalized, basal OCR from stress tests of Nos2−/− macrophages treated with vehicle or FA 1 h prior to O.N. stimulation. Data (n = 3) were analyzed by two-way ANOVA with Sidak’s post-tests. All error bars display mean ± SEM. Source data are provided as a Source Data file.
