Establishment and characterization of HOX11-immortalized hematopoietic cell lines. HOX11 constructs were transduced into murine BM progenitors, and cells were maintained in IL-3–containing media until lines were established (> 3 months in culture). Transduction with each construct was performed at least 3 times, with the exception of D1 and M5 (n = 2). Wright-Giemsa–stained cells generated with HOX11 WT (A), D6 (B), and Thr47Ile (C). Original magnification, × 100. (D) Western blot analysis of HOX11 protein expression in representative BM progenitor-derived cell lines using either anti-HOX11 polyclonal antibody (WT, D1, M1, M2, M3, and Thr47Ile mutants) or anti-FLAG antibody (D6 mutant). (E) Southern blot analysis of proviral copy number in 3 independently derived HOX11 WT and HOX11 D6 cell lines. Genomic DNA (10 μg), isolated from 107 cells and digested with EcoRI, was resolved on a 1% agarose gel and hybridized with a neo probe. (F) HOX11 WT cells represent a bipotential monocytic-granulocytic precursor. Three HOX11 WT and HOX11 D6 cell lines were treated with phorbol myristate acetate (PMA; 100 ng/mL). After 72 hours, surface expression of the granulocytic marker Gr-1 and the macrophage marker Mac-1 was determined by immunofluorescence flow cytometric analysis.
