E76K SHP-2 expression enhances proliferation and perturbs differentiation of fetal liver cell growth in liquid cultures. (A) GFP+ cells that had been transduced with WT or E76K SHP-2 were isolated by sorting and plated in quadruplicate at 1.2 × 106 cells/well in medium containing 15% FBS and 2 ng/mL GM-CSF. GM-CSF was removed from the culture medium after 48 hours (on day 3), and live cells were counted every other day. Error bars reflect standard error of the mean. (B) Cytospin preparations of cells removed after 5 days in culture (original magnification, × 200). Whereas most of the cells in WT cultures are mature neutrophils, E76K SHP-2 cultures show a predominance of monocyte-macrophage cells with some blastlike elements. (C) Progenitor colony growth of cells isolated from WT and E76K SHP-2 liquid cultures in methylcellulose medium supplemented with IL-3, IL-6, SCF, and EPO. (D) BrdU incorporation by liquid culture cells maintained in GM-CSF over time. Data shown are for 2 hours of labeling. Similar differences were observed in WT and E76K SHP-2 cultures that were labeled for 4 hours. (E) Flow cytometry analysis of BrdU incorporation by WT and E76K SHP-2 liquid cultures on day 6. All of the data shown in panels A-E are representative of 3 independent experiments. The numbers on the plots indicate the percentage of BrdU-positive cells.
