Itaconate impairs SEP proliferation in a Nrf2-dependent manner. (A) Nrf2 protein analysis in Kit⁺ SEPs (left) and stromal cells (right). Protein levels relative to Hsp70 were calculated using ImageJ (n = 3 per time point; unpaired t test). Corresponding western blots are shown in supplemental Figure 2E. (B) Analysis of total SEP counts in WT and Nrf2^–/– SEEM cultures on days 3 and 5 (n = 5 per group; unpaired t test). (C) Flow cytometry quantification of the percentages (top) and absolute numbers (bottom) of indicated populations of SEPs in WT and Nrf2^–/– SEEM cultures on day 5 (n = 4 per group; unpaired t test). (D-F) WT and Nrf2^–/– SEEM cultures were treated with or without 125-μM OI for 3 days. qRT-PCR analysis of Nqo1 expression (D), analysis for numbers of Kit⁺Sca1⁺ SEPs (E), and qRT-PCR analysis of Nos2 expression (F) (n = 4; 2-way ANOVA/Fisher least significant difference). CTL, control; Hsp70, heat shock protein 70; K⁺S⁺34⁺133⁺, Kit⁺Sca1⁺CD34⁺CD133⁺; n.s., not significant.