G-CSF restores cytokine signaling and promotes survival of Nrf2^−/− HSPCs. (A) Gene expression of inflammatory cytokines, cytokine receptors, and prosurvival molecules is down-regulated in Nrf2^−/− KSL cells. BM was harvested from 5 mice/group and KSL cells were isolated by FACS. RNA was isolated from sorted cells and quantitative RT-PCR performed for specific genes. Data are average values from 2 separate experiments and are normalized to expression levels in Nrf2^+/+ KSL cells (represented by the dotted line). BCL2A1 inidicates BCL2-related protein A1; CCL2, chemokine (C-C motif) ligand 2; CCL5, chemokine (C-C motif) ligand 5; CSF1, colony-stimulating factor 1 (macrophage); CSF1R, colony-stimulating factor 1 receptor; CXCL10, chemokine (C-X-C motif) ligand 10; IL1B, interleukin 1 beta; IL1R, interleukin 1 receptor type II; IL10, interleukin 10; and TREM1, triggering receptor expressed on myeloid cells 1. (B) G-CSF treatment leads to expansion of KSL, LT-HSC, and ST-HSC compartments in both Nrf2^+/+ and Nrf2^−/− mice. Mice were treated with 100 μg/kg of G-CSF daily or vehicle control for 1 week, and BM was isolated and labeled with Abs and analyzed by flow cytometry. Data are averages from 3 mice per group. (C) G-CSF treatment enhances survival of Nrf2^+/+ and Nrf2^−/− HSPCs. Mice were treated with 100 μg/kg of G-CSF daily or vehicle control for 1 week. BM from 3 mice in each group was isolated, pooled, and treated with H₂O₂ (50μm for 30 minutes) or control and cultured in serum-free medium at an atmospheric O₂ concentration for 6 hours. Viable cells were annexin V⁻/propidium iodide⁻. Data are averages from 3 replicates. (D) G-CSF treatment corrects many of the gene-expression differences seen between Nrf2^+/+ and Nrf2^−/− KSL cells. Mice were treated with 100 μg/kg of G-CSF daily or control for 1 week, and KSL cells were isolated from BM. Quantitative RT-PCR was performed and analyzed as described in panel A.