Figure 2
Figure 2. Serial transplantation assays show that Cul4A+/− HSCs have a self-renewal defect. (A) Secondary transplantation with donors from the 1:1 primary transplantation. For the primary transplantation with a 1:1 ratio of test to competitor cells, one recipient of Cul4A+/− test cells and a wild-type control with similar percentage donor chimerism 6 months after transplantation were used as donors in secondary transplantations. For each donor, 106 bone marrow LDMNCs were transplanted into 3 to 4 lethally irradiated wild-type recipients, and overall percentage donor chimerism was measured 1 to 6 months after transplantation for Cul4A+/− (■) and wild-type (□) test cells, similar to that described in Figure 1B. *P ≤ .02. (B) Engraftment into myeloid and lymphoid lineages. For the secondary transplantation described in panel A, engraftment of Cul4A+/− (filled bars) or wild-type (open bars) test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was determined 5 months after transplantation as described in Figure 1C. *0.002 ≤ P < .05. (C) Tertiary transplantations. Tertiary transplantations were performed with donors from the secondary transplantation described in panels A and B: a recipient of Cul4A+/− test cells and a corresponding wild-type test cell recipient with closely matched percentage donor chimerism 6 months after transplantation were donors. For each donor, 106 bone marrow LDMNCs were transplanted into each of 5 to 6 wild-type recipients. Overall percentage donor chimerism was determined (as described in Figure 1B) 1 to 6 months after transplantation for Cul4A+/− (■) and wild-type (□) test cells. *P < .006. (D) Tertiary transplantation; engraftment into myeloid and lymphoid lineages. For the tertiary transplantation described in panel C, engraftment of Cul4A+/− (filled bars) or wild-type (open bars) test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was determined 6 months after transplantation as described in Figure 1C. *P < .007. The difference between Cul4A+/− and control engraftment into the T-cell lineage was not quite significant (P = .054). However, the difference in engraftment into this lineage is clearly statistically significant when fewer cells were transplanted (F). (E) Secondary transplantation with donors from the 1/3:1 primary transplantation. For the primary transplantation with a 1/3:1 ratio of test to competitor cells, 2 recipients of Cul4A+/− test cells and, for each, a wild-type control with similar percentage donor chimerism were used as donors in secondary transplantations. For each donor, 106 bone marrow LDMNCs were transplanted into 3 to 4 recipients, and overall percentage donor chimerism was measured 1 to 6 months after transplantation for Cul4A+/− (■) and wild-type (□) test cells as described in Figure 1B. *P ≤ .007. (F) Secondary transplantation with 1/3:1 donors; engraftment into myeloid and lymphoid lineages. For the secondary transplantation described in panel E, engraftment of Cul4A+/− (filled bars) or wild-type (open bars) test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was determined 6 months after transplantation as described in Figure 1C. *.001 ≤ P ≤ .02. For all graphs, mean (± SEM) is graphed.

Serial transplantation assays show that Cul4A+/− HSCs have a self-renewal defect. (A) Secondary transplantation with donors from the 1:1 primary transplantation. For the primary transplantation with a 1:1 ratio of test to competitor cells, one recipient of Cul4A+/− test cells and a wild-type control with similar percentage donor chimerism 6 months after transplantation were used as donors in secondary transplantations. For each donor, 106 bone marrow LDMNCs were transplanted into 3 to 4 lethally irradiated wild-type recipients, and overall percentage donor chimerism was measured 1 to 6 months after transplantation for Cul4A+/− (■) and wild-type (□) test cells, similar to that described in Figure 1B. *P ≤ .02. (B) Engraftment into myeloid and lymphoid lineages. For the secondary transplantation described in panel A, engraftment of Cul4A+/− (filled bars) or wild-type (open bars) test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was determined 5 months after transplantation as described in Figure 1C. *0.002 ≤ P < .05. (C) Tertiary transplantations. Tertiary transplantations were performed with donors from the secondary transplantation described in panels A and B: a recipient of Cul4A+/− test cells and a corresponding wild-type test cell recipient with closely matched percentage donor chimerism 6 months after transplantation were donors. For each donor, 106 bone marrow LDMNCs were transplanted into each of 5 to 6 wild-type recipients. Overall percentage donor chimerism was determined (as described in Figure 1B) 1 to 6 months after transplantation for Cul4A+/− (■) and wild-type (□) test cells. *P < .006. (D) Tertiary transplantation; engraftment into myeloid and lymphoid lineages. For the tertiary transplantation described in panel C, engraftment of Cul4A+/− (filled bars) or wild-type (open bars) test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was determined 6 months after transplantation as described in Figure 1C. *P < .007. The difference between Cul4A+/− and control engraftment into the T-cell lineage was not quite significant (P = .054). However, the difference in engraftment into this lineage is clearly statistically significant when fewer cells were transplanted (F). (E) Secondary transplantation with donors from the 1/3:1 primary transplantation. For the primary transplantation with a 1/3:1 ratio of test to competitor cells, 2 recipients of Cul4A+/− test cells and, for each, a wild-type control with similar percentage donor chimerism were used as donors in secondary transplantations. For each donor, 106 bone marrow LDMNCs were transplanted into 3 to 4 recipients, and overall percentage donor chimerism was measured 1 to 6 months after transplantation for Cul4A+/− (■) and wild-type (□) test cells as described in Figure 1B. *P ≤ .007. (F) Secondary transplantation with 1/3:1 donors; engraftment into myeloid and lymphoid lineages. For the secondary transplantation described in panel E, engraftment of Cul4A+/− (filled bars) or wild-type (open bars) test cells into the granulocyte, macrophage, B-cell, and T-cell lineages was determined 6 months after transplantation as described in Figure 1C. *.001 ≤ P ≤ .02. For all graphs, mean (± SEM) is graphed.

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