HSC depletion of myr-AKT–transduced BM is dependent on mTOR signaling but not on increased ROS. (A) Rapamycin but not N-acetylcysteine (NAC) rescues cobblestone-forming activity of myr-AKT–transduced BM in the LTC-IC assay. A total of 500 000 retrovirally transduced GFP-sorted BM cells/well were cocultured for 4 weeks with 300 000 OP9 stromal cells. After 4 weeks in coculture, cells were trypsinized and plated into M3434 methylcellulose media, and colonies were scored after 7 days. Rapamycin (Rap) or NAC was added at the time of initial plating, and treatment was continued for 4 weeks. Each drug experiment was performed with at least 5 replicates. (B) ROS levels in the LSK compartment after short-term induction of AKT signaling in vivo. Induction of myr-AKT expression using the myr-AKT-ER BMT system was performed as previously described. Thy1.1⁺ LSK and progenitor cells were sorted from the BM, and freshly sorted cells were stained with DCF-DA to determine relative levels of ROS. The experiment was performed twice, with 3 or 4 mice per group in each experiment. Right bar graphs: Fold change in DCF-DA peak intensity of sorted LSK and progenitor cells. The values for vector samples were normalized to 1 for the analysis.