Intact HOXB4 homeodomain is essential for HOXB4-induced expansion of primitive BM cells. (A) Expansion of GFP⁺ cells in liquid cultures initiated with 10% GFP⁺ (control, or HOXB4(N→A)⁺, or HOXB4(WT)⁺) cells, and 90% of nontransduced competitors. ▪ indicates HOXB4(WT); ▴, HOXB4(N→A); and ○, control GFP. Results represent mean values ± the standard deviation (SD) of a representative experiment (n = 3) performed in duplicate. (B) In vitro expansion over a 10-day period of myeloid CFCs derived from indicated sorted populations of transduced BM cells. Results represent mean values ± SD of a representative experiment (n = 2) performed in quadruplicate cultures. (C) Southern blot analyses of proviral integrations in DNA isolated from bone marrow (B), spleen (S), and thymus (T) of primary recipients, and the secondary recipients of HOXB4(WT). Individual recipients and the numbers of transplanted cells are shown on top, and the proportions of transplant-derived Ly5.1 cells in these recipients are listed bellow each lane. DNA was digested with KpnI, which releases the integrated provirus. Probes are listed to the right. Erythropoietin receptor (EpoR)–derived signal is representative of DNA loading. Southern blot analyses of proviral integrations in DNA isolated from hemopoietic tissues of primary and secondary HOXB4(N→A) (D) and neo^r recipients (E) were performed as described for panel C. (F) Total CRU recovery following competitive reconstitution with neo^r-, HOXB4(N→A)-, or HOXB4(WT)-transduced BM cells determined for each group of recipients (mean ± 95% CI). □ indicates neo^r recipients; ▦, HOXB4(N→A) recipients; and ▪, HOXB4(WT) recipients.