RUNX1 inhibits erythroid differentiation. (A) Scheme of murine MEP preparation. MEPs were harvested from bone marrow of adult mice and purified by FACS. Colonies from transduced cells (Venus⁺) were analyzed 8 days after seeding. N = 3 independent experiments. (B) RUNX1 decreases erythroid differentiation of MEPs. The frequency of erythroid colonies was decreased upon wild-type RUNX1 but not upon RUNX1mut expression. (C) RUNX1 enhances megakaryocytic (mega.) differentiation of MEPs. The frequency of megakaryocytic colonies was increased upon RUNX1 expression, whereas RUNX1mut did not show this effect. Colonies with more than 10 cells were included. (D) CFU assay with hCD34⁺ cells transduced with a RUNX1 expression vector. Sorted (green fluorescent protein–positive) cells were subjected to a CFU assay. The relative frequency of erythroid colonies was decreased on RUNX1 expression compared with the control. N = 4 independent experiments. (E) The percentage of benzidine-positive (heme-expressing) cells was determined using resuspended cells from the CFU assay. (F) RUNX1 inhibits erythroid differentiation of hCD34⁺ cells in suspension culture. hCD34⁺ cells were transduced with an empty vector or a RUNX1 expression vector. Cells were kept in suspension culture under conditions that allow erythroid differentiation. Erythroid surface markers were measured by FACS. The percentage of cells expressing the erythroid surface marker GYPA was reduced in RUNX1-expressing hCD34 cells. (G) RUNX1 expression in K562 cells reduces cell surface expression of GYPA. Shown is the median fluorescence intensity (MFI) of GYPA-allophycocyanin staining in control cells and K562 cells transduced with RUNX1. (H) Knockdown of RUNX1 in hCD34⁺ cells using 2 different shRNAs reduced the RUNX1 mRNA amount. (I) RUNX1 knockdown led to an increased number of erythroid colonies in a CFU assay. (J) The knockdown led to an increased number of (heme-expressing) benzidine-positive cells. For RUNX1 knockdown, 2 different shRNAs were used, which were in distinct vector backbones. Control vectors expressed a nontargeting shRNA. The P values were calculated using Student t test. *P < .05; **P < .01; ***P < .001. mRNA, messenger RNA; RUNX1mut, DNA binding–deficient RUNX1 mutant.