Competitive transplantation of Notch-activated LT-HSCs from Rosa26-Flox-STOP-Flox-ICN-GFP mice leads to cell autonomous exhaustion of HSC activity and numbers. (A) Mx-cre^tgRosa26-Flox-STOP-Flox-YFP (“YFP control”) mice and Mx-cre^tgRosa26-Flox-STOP-Flox-ICN-GFP (“Notch”) mice were injected with pI-pC to activate YFP and ICN-GFP, respectively in HSCs. Then, 4 weeks later YFP⁺ or GFP⁺ Lineage⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻ BM cells (HSCs) were sorted and transplanted into syngeneic hosts at 500 cells per mouse along with 200 000 whole syngeneic BM cells. (B) BM DP T-cell production (CD4⁺CD8⁺) in YFP control and Notch mice was measured in the bone marrow at 4 and 16 weeks after transplantation. (C) Scatter plot analysis of DP T-cell production by YFP control and Notch mice at 4 and 16 weeks after transplantation. (D) The LT-HSC compartment (Lineage⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) was measured for donor reconstitution by YFP⁺ control and Notch HSCs at 4 and 16 weeks after transplantation. Scatter plot analysis of donor-derived (YFP⁺ or Notch) LT-HSCs (Lineage⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻) at 4 and 16 weeks after transplantation. (E) Cell-cycle analysis was performed in sorted YFP control and Notch LT-HSCs (YFP⁺ or GFP⁺ Lineage⁻Sca-1⁺c-Kit⁺CD150⁺CD48⁻ BM cells). Propidium iodide⁺ cells were excluded. Pyronin⁺/Hoechst 33 342⁺ cells (top left quadrant) are in G₁. Pyronin⁻/Hoescht⁻ cells (bottom left quadrant) are in G₀. Pyronin⁺/Hoechst 33 342⁺ cells (top right quadrant) are in S/G₂/M.