Expression of Ras in CD34⁺ cells and its effect on colony formation. (A) Expression of GFP and CD34 on retrovirally transduced cells (day 3); quadrants delineate autofluorescence/background staining using isotype-matched controls; figures represent the frequency of cells (%) in each quadrant. (B) Corresponding expression of mutant Ras (using a Ras^V12-specific antibody) and total Ras (using a pan-specific antibody). (C) Cells were plated for colony-forming assay by limiting dilution on day 3 or day 8 of culture (as indicated) in IL-3, GM-CSF, G-CSF, and SCF. Colonies were scored 7 days later. GFP positivity and lineage identity were determined by flow cytometric analysis (see “Materials and methods”). G indicates granulocytic; M, macrophage; GM, granulocyte/macrophage; U, colonies for which lineage could not be unambiguously defined by cytometric analysis. (D) Average colony size for each colony type (for colonies plated on day 3); error bars represent SD; *P ≤ .05. Each data set (C-D) represents the mean of at least 4 independent replicate experiments (n = 4).