EKLF knockdown in normal human progenitors enhances megakaryocytic differentiation at the expense of erythrocytic differentiation. Human cord blood CD34⁺ cells were amplified for 3 days in E/MK medium, infected for 3 hours with either Eklf shRNA or control SCR shRNA lentiviruses. Immediately after infection, CD36⁺CD31^Med cells enriched in bipotent progenitors were sorted by FACS and reseeded for 7 days in E/MK as described in “Lentivirus production and cell infection” and Figure S1. Each well was inspected under fluorescent microscope to identify clones containing GFP⁺ cells attesting successful infection and classified as megakaryocytic, erythrocytic, or mixed clones based on light-microscope observation, FACS analysis of the expression of GPA and CD41, and benzidine staining. Erythrocytic clones were identified by a large number of small cells expressing GPA but not CD41 and containing benzidine-positive cells; megakaryocytic clones were identified by a small number of large cells expressing CD41 but not GPA and containing no benzidine-positive cells; mixed clones were identified by an intermediate number of both types of cells. (A) Typical FACS diagrams and light-microscope fields illustrating erythrocytic, megakaryocytic, and mixed clones obtained. (B) Histogram shows the percentages of these 3 different types of GFP⁺ clones derived from cells infected with either Eklf shRNA (■) or control shRNA virus (□) (means and SD from 3 independent experiments with 60-92 GFP⁺ clones recorded for each condition). Statistically significant differences (P < .05; paired Student test) are indicated by asterisks (NS indicates nonsignificant).