Extended Data Fig. 8. Itaconate, but not citraconate, depresses mitochondrial respiration in LPS/IFNγ-activated dTHP1 cells.

LPS/IFNγ stimulation and treatment with citraconate or itaconate (1 mM or 25 mM) were performed as in Fig. 4m-o. Mitochondrial respiration was measured by Seahorse assay in unstimulated dTHP1 cells or after 12 h of stimulation. a-c, Bar graphs showing oxygen consumption rate (OCR) due to basal respiration, maximal respiration (a and b), and spare respiratory capacity (c). 25 mM itaconate significantly reduced maximal respiration and spare respiratory capacity in LPS/IFNγ-induced dTHP1 cells, whereas there was a tendency (p=0.076 and 0.096 respectively) of 1 mM citraconate to prevent this decline, and there was a tendency of 25 mM itaconate to normalize spare respiratory capacity (p=0.082). In a n (biological replicates) were as follows: untreated = 5; 1 mM Citra = 4; 25 mM Citra = 3; 1 mM Ita = 4; 25 mM Ita = 4. In b n were as follows: untreated = 5; 1 mM Citra = 5; 25 mM Citra = 3; 1 mM Ita = 4; 25 mM Ita = 4. Results in c are computed from the experiment shown in a and b and therefore have the same n as the respective treatments in a and b. Means ±SD; one-way ANOVA followed by Dunnett’s multiple comparisons test. P values: * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001. d-h, OCR output curves that form the basis of the graphs shown in a-c. d, Unstimulated vs. LPS/IFNγ-stimulated cells. e, Citraconate treatment, unstimulated cells. f, Citraconate treatment, LPS/IFNγ-stimulated cells. g, Itaconate treatment, unstimulated cells. h, Itaconate treatment, LPS/IFNγ-stimulated cells. Results in d-h are computed from the experiment shown in a and b and therefore have the same n as the respective treatments in a and b. Means ±SD. P values: * ≤0.05, ** ≤0.01, *** ≤0.001, **** ≤0.0001; one-way ANOVA with Dunnett’s multiple comparisons test. Abbreviations: FCCP = carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; Oligo = oligomycin; Rot = rotenone; AA = Antimycin A.