C/EBPδ suppresses growth of primary hematopoietic progenitors. (A) Colony assays showing the growth inhibitory effect of C/EBPδ on hematopoietic progenitor cells. Murine bone marrow cells were transduced with an ER-inducible C/EBPδ construct or empty vector as a control. After 48 hours of transduction, cells were puromycin selected for 2 days. Colony assays were performed in the presence of β-estradiol to activate C/EBPδ. The results are shown as the mean ± SD of 3 independent experiments. (B) Western blot analysis of the colonies obtained in panel A. Blots were probed with anti-C/EBPδ and antiactin antibodies to confirm ectopic expression of C/EBPδ in the colonies. (C) C/EBPδ-transduced primary bone marrow cells in colony assay after replating. C/EBPδ expression completely abolished the colony growth of primary cells on replating. Photograph of representative areas of the plates demonstrates the lack of colony growth after replating in C/EBPδ-overexpressing cells in the presence of β-estradiol. (D) The bars indicate the inhibitory effect of C/EBPδ on colony formation in replating experiments. Results show mean ± SD of triplicates of 2 independent experiments. (E) Western blot analysis with G-CSFR and c-myc antibodies in total protein lysate from C/EBPδ or empty vector control–transduced bone marrow cells. The blot was stripped and rehybridized with actin antibodies. (F) Wright-Giemsa–stained cytospin slides under light microscopy. C/EBPδ or the empty vector–transduced primary bone marrow cells were incubated with β-estradiol for induction of C/EBPδ. Representative morphologic changes seen at day 7 of β-estradiol treatment are shown.