miR-486-5p inhibition blocks erythroid differentiation and reduces growth of CB CD34⁺ cells. (A) The lentivirus vector miRZip-anti-miR-486-5p is shown. (B-F) CD34⁺ cells transduced with anti-miR-486-5p and control vector (n = 3) were selected by flow cytometry and cultured in GEMM medium for 6 days and the percentage of GPA, CD34, CD11b, CD33, and CD14 cells were analyzed by flow cytometry. Representative results of flow cytometry analysis of GPA and CD11b expression are shown in (B). The percentage of cells expressing these markers at day 3 (C) and day 6 (D) and the total cell number of expressing these markers at day 3 (E) and day 6 (F) are shown. (G) The number of CFU-GM and burst-forming unit-erythroid colonies generated from selected CD34⁺GFP⁺ cells after culture in methylcellulose progenitor assays. (H) CD34⁺GFP⁺ cells were labeled with Dye670 and cultured in HGF of SFEM medium for 3 days. Cell division was evaluated on the basis of reduction of Dye670 fluorescence intensity. A proliferation index was calculated using ModFit software. (I) CD34⁺GFP⁺ cells were cultured for SFEM medium with HGF combination or 48 hours and treated with Hoechst 33342 (2 µg/mL) for 1 hour. DNA content was analyzed by flow cytometry and cell-cycle distribution calculated using ModFit software. Cumulative results represent the mean ± SEM. *P < .05, **P < .01, ***P < .001.