Hemopoietic reconstitution and expansion of HSCs induced by HOXB4 are independent of its collaboration with PBX. (A) Flow cytometric analysis of BM, splenic, and thymic cell populations of mice that received transplants of comparable numbers of GFP⁺ control, HOXB4(W→G), or HOXB4(WT)-transduced BM cells. Proportions of GFP⁺ cells in myeloid (Mac-1) and lymphoid (B-220 and CD4/CD8) populations were determined for recipients killed 12 weeks after transplantation. Results shown represent mean values ± SD determined for 4 mice analyzed in each group of recipients. (B-C) CFC content (B) and distribution (C) in BM and spleen of recipients described for panel A. Colonies were identified as containing predominantly granulocytes (Gr), granulocytes and macrophages (GM), macrophages (Ma), erythrocytes (Er), or a mixture of these cells together with megakaryocytes (Mix). (D) Top panel: representative Southern blot analysis of proviral integration patterns in hemopoietic tissues isolated from primary recipients of control GFP, HOXB4(W→G), or HOXB4(WT)-transduced BM cells presented in panel A. DNA was digested with EcoRI, which cuts once within the integrated provirus, such that each band represents a unique integration event on blots probed with GFP. Bottom panel: Western blot of HOXB4 levels in bone marrow, spleen, and thymus of recipients identified in panel D. Tubulin levels are shown as control for protein loading and transfer. (E) HSC regeneration in recipients of HOXB4(W→G) or HOXB4(WT)-transduced BM cells. For each group of recipients, results represent mean values ± 95% CI determined for 2 primary recipients.