Overexpression of D171N promotes inhibition of differentiation and increase in self-renewal capacity. (A) Pictogram of pMXs.IG retroviral constructs of the FLAG-tagged RUNX1 WT and the D171N mutant (D171N). The difference in cDNA sequence of the mutant from the WT is indicated by an arrowhead. LTR, long terminal repeat. (B) Human CD34⁺ CB cells were transduced with the indicated vector. A typical flow cytometry profile of cells retrovirally transduced with pMXs.IG, WT, or D171N shows the transduction efficiency. The GFP⁺ cells shown within the gate were collected. (C) Anti-FLAG immunoblotting of sorted GFP⁺ cells confirmed the expression of FLAG-tagged RUNX1 proteins. Anti–β-actin antibody was used as control. (D-H) Ten thousand cells were plated in methylcellulose culture dishes. GEMM, colony-forming unit–granulocyte, erythroid, macrophage, megalocyte. Data are expressed as mean ± SD of 6 independent experiments and compared with control (pMXs.IG). *P < .05; **P < .01. (D) Colony numbers were counted after 14 days. (E) Photomicrographs (×40) of representative colonies found in the plates with an IX71 microscope and a DP12 camera (Olympus). (F) The cell number per colony was calculated by total GPA⁺ cells/total BFU-E colonies and total CD13⁺ cells/total CFU-GM colonies. (G) GFP⁺ cells were analyzed by flow cytometry for the indicated surface markers. (H) Colony number and cell proliferation fold in CFC replating assay. (I) LTC-IC assay in bulk was carried out in duplicate, and the average number of LTC-IC per 10 000 original input cells and SD of 4 independent experiments are indicated. **P < .01.