Figure 2. AMD3100 fails to mobilize PCs that express a constitutively-active c-kit mutant (c-kit^D816V).

(A) WT BM MNCs were isolated, transduced ex vivo with retroviral vectors coding for eGFP-linked WT c-kit (MSCV-HyKIT^WT-IRES-eGFP [MIG-HyKIT^WT]) or eGFP-linked constitutively active c-kit (MSCV-HyKIT^D816V-IRES-eGFP [MIG-HyKIT^D816V]), and transplanted (independently) into WT mice after radiative ablation of the endogenous BM. Three weeks after BM transplantation, the recipient mice were subcutaneously injected with AMD3100 (5 mg/kg) (+) or PBS (−); 2 hours later, PB and BM MNCs were isolated, and the numbers of PCs transduced with WT c-kit or constitutively active c-kit (i.e., the number of GFP⁺ PCs) in the PB and BM were quantified via the colony-forming assay. (B) Flow cytometry analysis for eGFP expression in HyKIT^WT- or HyKIT^D816V-transduced BM MNCs before transplantation. (C) The number of BM MNCs and the proportions of BM and PB MNCs in recipient mice that expressed GFP three weeks after BM transplantation. (D-E) The number of HyKIT^WT- or HyKIT^D816V-transduced colony-forming PCs (i.e., GFP⁺ colonies) in the PB (D) and BM (E) of mice treated with AMD3100 or PBS. Values are mean ± SEM; n=10 per group.