Itaconate inhibits NO-dependent proliferation of SEPs. (A) SEPs were treated with or without 125-μM OI at indicated days of SEEM cultures. On day 5 of SEEM cultures, total SEP cell counts were measured (n = 4 per group; 1-way analysis of variance [ANOVA]/Dunnett test). (B) SEPs were treated with or without 125-μM OI on SEEM day 3 for 48 hours, followed by flow cytometry analysis of SEPs. A representative flow cytometry plot shows pregated Kit⁺Sca1⁺ cells with additional markers CD34 and CD133. (C) Quantification of percentages (left) and absolute number (right) of the indicated populations shown in panel B. (n = 4 per group; unpaired t test). (D-F) SEPs were treated with or without 125-μM OI on SEEM day 3 for 48 hours. (D) Quantification of intracellular NO levels in Kit⁺Sca1⁺ SEPs by mean fluorescence intensity (MFI) of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate staining (left). Analysis of NO MFI in the indicated SEP populations (right; n = 3 per group; unpaired t test). (E) quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of Nos2 mRNA expression in SEPs on day 5 of SEEM culture treated with or without OI (n = 4; unpaired t test). (F) Nos2 expression in stromal cells. SEP cultures were treated with OI as indicated above. On days 4 and 5 of SEEM culture, stromal cells were analyzed by flow cytometry for Nos2 expression and markers for monocytes and macrophages as indicated (n = 3 per time point; paired t test). (G) SEEM cultures were treated with 125-μM OI alone or in combination with SNAP at the indicated concentrations for 24 hours. Flow cytometry quantification of numbers of Kit⁺Sca1⁺ SEPs (n = 3 per group; 1-way ANOVA/Tukey test). (H) WT and Irg1^–/– SEPs were cultured in SEEM for 5 days. qRT-PCR analysis Nos2 mRNA expression (left) and flow cytometry quantification (right) of absolute numbers of indicated populations of SEPs (n = 4 per genotype; unpaired t test). Data represent mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. K⁺S⁺34⁺133⁺, Kit⁺Sca1⁺CD34⁺CD133⁺; SNAP, S-nitroso-N-acetyl-DL-penicillamine.