Effect of Egr-1 on differentiation of stem cell–enriched BM cells stimulated for erythroid, macrophage, and granulocyte differentiation by IL-3 plus Epo.
(A) Effect of Egr-1 on differentiation of 5-FU BM cells stimulated with IL-3 plus Epo. After infection of 5-FU BM cells with MSCVneo (BMneo, ░) or MSCVneo Egr-1 (BMEgr-1, ▨), cells were assayed in methylcellulose supplemented with IL-3 (3% WEHI-3B–conditioned medium) and Epo (10 U). Cells were seeded at concentrations of 0.5 × 105 cells/mL in 35-mm tissue-culture dishes in the presence of G418 (650 μg/mL). Colonies generated after 8 days in the methylcellulose were isolated, pooled, washed, and resuspended in 1 × PBS, and cytospin smears were prepared. To determine different cell types, at least 300 May-Grünwald–stained cells were scored for each sample. The percentage of erythroid cells was also determined by benzidine staining. (B) Representative photomicrographs of May-Grünwald–stained cytospin smears of 5-FU BM cells obtained from 8-day colonies in methylcellulose with IL-3 plus Epo that were generated by uninfected BM cells (control BM without G418) and cells infected with MSCVneo (BMneo plus G418) or MSCVneo Egr-1 (BMEgr-1 plus G418).
