We isolated bone marrow (BM) cells

We isolated bone marrow (BM) cells from young (2 months old) and old (26 months old) C57BL/6 mice and analyzed them for surface markers to identify HSCs and for intracellular staining of phosphorylated mTOR (p-mTOR). Using flow cytometry, we found that the amount of p-mTORwas significantly increased in both HSC-enriched Lin− Sca-1+ c-Kit+ (LSK) and the Flk2− lin−Sca-1+ c-kit+ CD150+ CD48− CD34− (FLSKCD150/48/34) HSCs from old mice compared to that in HSCs (Fig. 1A) from young mice. Consistent with the increase in phosphorylated mTOR, the abundance of the phosphorylated form of the mTOR complex 1 (mTORC1) substrate S6K and of the S6K substrate S6 was significantly increased in HSCs from old mice compared to that in HSCs from young mice (Fig. 1,B and C). These data indicate that the overall activity of mTOR in HSCs from old mice is greater than that in HSCs from young ones. To see whether this increase in mTOR phosphorylation was secondary to increased activity in the phosphoinositide 3-kinase (PI3K)–AKT signaling pathway,we evaluated AKT activation by measuring the abundance of AKT phosphorylated on Ser residue 473 (p-AKT) by flow cytometry. We found that the amount of p-AKT in HSCs from young and old mice was indistinguishable (Fig. 1D).