Increased itaconate production during differentiation alleviates NO-dependent erythroid inhibition. (A) SEPs were harvested from WT or Irg1^–/– cultures on SEEM day 5 and SEDM day 3. Western blot analysis of Nos2 protein expression, with β-actin as a loading control. (B) Analysis of Nos2 protein expression in WT control, Irg1^–/–, and Irg1^–/– plus125-μM OI on day 3 of SEDM culture (left); β-actin is the loading control; Nos2 protein levels (right) calculated relative to β-actin calculated using ImageJ (n = 4; unpaired t test). (C) Flow cytometry analysis of NO MFI from WT control, Irg1^–/–, and Irg1^–/– plus 125-μM OI, analyzed on day 3 of SEDM culture (n = 4; unpaired t test). (D-F) SEPs isolated from WT SEDM cultures at day 3 were compared with Irg1^–/– SEDM cultures treated with or without 1400W for 3 days; stress BFU-E (D), percent Kit⁺Sca1^– SEPs on SEDM day 3 (E), and mRNA expression (F) of select erythroid genes, EpoR, Cpox, and β-major globin (Hbb-b1; n = 4 per group; 1-way ANOVA/Tukey test). n.s., not significant.