Itaconate levels increase during the transition to differentiation. (A) SEPs were isolated from SEEM cultures on days 1 and 3 for metabolomics analysis. Volcano plot shows the changes in metabolites between day 1 and day 3 SEPs in SEEM (n = 5 per time point). (B) Levels of itaconate relative to spike in chlorpropamide were calculated and normalized to SEEM day 5 (EM D5 = 1; n = 5 per time point). (C) Expression of Irg1 mRNA (left) and protein (right) in Kit⁺ SEPs (top). Expression of Irg1 mRNA (left) and protein (right) stromal cells (bottom). Relative Irg1 mRNA expression was normalized to 18S ribosomal RNA. Irg1 Protein was normalized to β-actin levels using ImageJ software (n = 3 per time point). Corresponding western blots are shown in supplemental Figure 1A. Data represent mean ± standard error of the mean (SEM). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ADP, adenosine diphosphate; CDP, cytidine diphosphate; dAMP, deoxyadenosine monophosphate; dCDP, deoxycytidine diphosphate; dCMP, deoxycytidine monophosphate; DM, differentiation media; EM D5, expansion media day 5 of culture; NA, not altered.