Loss of Mysm1 drives HSC from quiescence to rapid proliferation. (A-B) Quiescence of HSC was evaluated with Hoechst 33258/Pyronin Y staining in the BM of WT and Mysm1^−/− mice. WT or Mysm1^−/− BM cells were stained for HSC surface antigens followed by Hoechst 33258/Pyronin Y staining. Representative FACS plots (left) of cells depicting G₀ (bottom left quadrant), G₁ (top left quadrant), and S/G₂/M (top right quadrant) in (A) LSK cells, and (B) LSK subsets. Bar graph (right) shows the percentage of cells in G₀ phase for each individual subpopulation. (C) WT, Mysm1^−/− mice received 2 mg of BrdU intraperitoneally daily for 5 days. Incorporation of BrdU was analyzed by FACS in BM LSK and its subsets. (D) FACS plots of cell cycle kinetics of WT and Mysm1^−/− LSK cells. (E) Cell-cycle analysis in CD150⁺CD48⁻LSK and CD34⁻Flt3⁻LSK cells. (F) Real-time PCR analysis of cyclins, cyclin-dependent kinases (Cdks), and Cdk inhibitors in LSK cells. The Ct values are normalized to glyceraldehydes-3-phosphate dehydrogenase and are presented relative to WT (value set as 1). (A-F) Data are representative of three independent experiments. *P < .05; **P < .01.