Secondary engraftment potential of Id1−/− bone marrow. For all panels, ▩ indicates wild-type and ■ indicates Id1−/− graft contributions. Secondary transplantations were conducted as described for Figure 3, except parallel cohorts of mice received either wild-type or Id1−/− marrow. Cells for each secondary transplantation experiment were pooled from at least 3 primary donors per genotype. Primary and secondary hosts were treated prior to transplantation with 6.5 Gy radiation. Evaluation of bone marrow chimeras 16 days after transplantation by immunofluorescent staining and flow cytometry showed that primary bone marrow was more than 40% donor derived. Marrow was pooled from at least 4 primary hosts before transplanting 2 × 106 cells per each secondary host. Secondary engraftment was evaluated 5 to 12 weeks later, as noted for each dataset. Engraftment analysis was also conducted as described for Figure 3. (A) Secondary engraftment from WBM transplantations. Left panel shows frequencies of host meeting myeloid (“M”), B-cell (“B”), or thymocyte (“T”) engraftment thresholds for each graft genotype 5 weeks after secondary marrow transplantation. Right panels give average donor-derived Ly6G+ (“M”) and CD19+ (“B”) cell numbers per femur, and donor-derived CD4+ and/or CD8+ cells per thymus (“T”). Error bars indicate SEM. Data were pooled from 5 replicate experiments where 25 mice received transplants per secondary marrow genotype. (B) Secondary engraftment from LSK-Thy1.1Lo enriched HSCs. In 2 replicate experiments, 103 LSK-Thy1.1Lo cells from either wild-type or Id1−/− donors were transferred to primary hosts, which served as donors for secondary grafts 16 days later. Top panels show average donor cells per femur observed from experiment 1, while bottom panels give results from experiment 2. Left panels show Ly6G+ myeloid engraftment at both 5 and 12 weeks after transplantation. Right panels show donor CD19+ B cells for the same time points. A minimum of 14 secondary hosts were evaluated at each time point per genotype.
