Effect of Egr-1 on clonogenicity and macrophage/granulocyte differentiation of stem cell–enriched BM cells.
(A) Infection efficiency of 5-FU BM cells with pMSCVneo. The 5-FU BM cells were cocultivated for 4 days with Bosc23 cells transfected with MSCVneo or MSCVneo Egr-1 retroviral-expression constructs. After infection, control and infected BM cells were assayed in methylcellulose supplemented with IL-3 (10% WEHI-3B–conditioned medium), SCM (10%), G-CSF (100 ng/mL), GM-CSF (100 ng/mL), or M-CSF (10% L-cell–conditioned medium as a source of M-CSF). Cells were seeded at concentrations of 0.5 × 105 cells/mL in 35-mm tissue-culture dishes in the presence or absence of G418 (650 μg/mL), and colonies were scored after 8 days. Values are mean (± SD) results from 3 independent experiments. (B) Effect of Egr-1 on clonogenicity of stem cell–enriched BM cells. After infection of 5-FU BM cells with either MSCVneo (BMneo) or MSCVneo Egr-1 (BMEgr-1), the cells were assayed in methylcellulose supplemented with the indicated cytokines and G418 (650 μg/mL) as described above. Colonies were scored after 8 days. Values are mean (± SD) results from 3 independent experiments. (C) Effect of Egr-1 on differentiation of stem cell–enriched BM cells. Uninfected bone marrow cells (BM control) or cells infected with MSCVneo (BMneo) or MSCVneo Egr-1 (BMEgr-1) were seeded in a methylcellulose culture supplemented with the indicated cytokines with or without G418, as described above. Colonies generated after 8 days were isolated and pooled, and cytospin smears were prepared and stained with May-Grünwald stain. To determine cell types, at least 300 May-Grünwald–stained cells were scored. Values are percentages (± SD) of cell types from 3 independent determinations.
