Nras^G12D/+hyperactivates ERK1/2 in HSCs and downregulation of the MEK/ERK signaling attenuates Nras^G12D/+HSC phenotypes. (A-B) Total BM cells from control or Nras^G12D/+ mice were enriched for Sca1⁺ cells. CD150⁺ CD41⁻ cells (enriched for HSCs) and CD150⁻ CD41⁻ cells (enriched for MPPs) were subsequently sorted from Sca1⁺-enriched cells. (A) Sorted cells were serum- and cytokine-starved for 30 minutes at 37°C. (B) Interleukin-3 (IL-3) stimulation was performed for 10 minutes at 37°C after starvation. Levels of phosphorylated ERK1/2 and AKT were measured using phospho-specific flow cytometry. HSCs (defined as [Lin CD48]^−/low cKit⁺ cells from sorted CD150⁺ CD41⁻ cells) and MPPs (defined as [Lin CD48]^−/low cKit⁺ cells from sorted CD150⁻ CD41⁻ cells) were gated for data analysis. To quantify the activation of ERK1/2, median intensities of pERK1/2 at different IL-3 concentrations are compared with their respective control cells at 0 ng/mL, which is arbitrarily set at 1. (C) Total BM cells were serum- and cytokine-starved for 1 hour and stimulated with or without 10 ng/mL of granulocyte macrophage colony-stimulating factor (GM-CSF) at 37°C for 10 minutes. AZD6244 or vehicle was mixed with cells for 30 minutes before GM-CSF stimulation. Levels of pERK1/2 or pSTAT5 were measured using phospho-specific flow cytometry. Non-neutrophil Lin⁻ c-Kit⁺ cells were gated for data analysis. Results obtained from one representative experiment are shown. (D) Quantification of BM and SP HSCs from control and Nras^G12D/+ (G/+) mice treated with vehicle or AZD6244 for 7 days. (E) Quantification of BM and SP common myeloid progenitors (CMPs) from control and Nras^G12D/+ (G/+) mice treated with vehicle or AZD6244 for 7 days. (F) EdU incorporation to quantify proliferating HSCs and WBM in control, Nras^G12D/+, Erk2^+/−, and Nras^G12D/+; Erk2^+/− mice. Data are presented as mean + standard deviation. WT, wild-type. *P < .05; **P < .01; ***P < .001.