Nrf2^−/− HSPCs have no increase in basal ROS but are more susceptible to oxidative stress. (A) ROS levels in Nrf2^+/+ and Nrf2^−/− BM cells. BM isolated from Nrf2^+/+ and Nrf2^−/− mice was incubated with H₂-CM-DCFDA to detect intracellular ROS by flow cytometry. Mean fluorescence intensity was measured in LT-HSC (CD34⁻ c-Kit⁺Sca1⁺Lin⁻), ST-HSC (CD34⁺ c-Kit⁺Sca1⁺Lin⁻), and Lin⁺ cells. Relative MFIs are normalized to wild-type LT-HSCs. Data represent mean values from 13 mice of each genotype in 4 different experiments. (B) Induction of intracellular ROS in Nrf2^+/+ and Nrf2^−/− BM cells by increasing doses of H₂O₂. BM cultured in normoxia for 30 minutes with H₂O₂ (50-200μM) to induce oxidative stress. BM from 3 mice per genotype was pooled for each concentration of H₂O₂. Data are mean fluorescence intensity values of H₂-CM-DCFDA (arbitrary units). (C) Nrf2^−/− progenitors show increased sensitivity to oxidative stress in vitro. Whole BM cells were treated with 50μM H₂O₂ for 30 minutes, plated in methylcellulose medium supplemented with cytokines, and colonies were scored at 14 days. Data are mean values from 3 individual mice plated in duplicated. *P = .012. (D) Schema for testing in vivo sensitivity of Nrf2^−/− LT-HSCs to radiation injury. Mice were transplanted with Nrf2^+/+ or Nrf2^−/− and allowed to form stable chimeras. Mice were then treated with a sublethal dose of radiation (400 rads) and their chimerism compared 4 months later to assess the effect of radiation on LT-HSCs. (E) Nrf2^−/− LT-HSCs show increased radiosensitivity. Four months after radiation, peripheral blood chimerism returned to baseline in mice transplanted with Nrf2^+/+ BM, but remained much lower in Nrf2^−/− chimeras, suggesting a selective loss of Nrf2^−/− LT-HSCs. *P = .018