MiR-24 inhibits activin A-induced erythroid differentiation of CD34⁺ hematopoietic progenitor cells. (A-D) CD34⁺ HPCs were transfected with GFP and miRNA plasmids, the plasmid expressing ALK4 that lacks the 3′-UTR or anti-miRNA oligoribonucleotides as indicated. Then GFP-positive cells were sorted and placed for colony-forming cell assays in semisolid media. CFU-E was scored at day 7 (A,C) of culture and BFU-E at day 14 (B,D). The data represent the mean plus or minus SD of 3 independent experiments. The asterisks indicate a significant difference between control miRNA and miR-24 or between anti-24 and its mutant control (*P < .05; **P < .01). (E) CD34⁺ HPCs were maintained in liquid erythroid differentiation culture condition for 7 days. MiR-24 expression at the indicated times was detected by stem-loop RT-PCR, and endogenous ALK4 expression was detected by RT-PCR. (F,G) CD34⁺ HPCs were transfected with GFP and miRNA vectors or anti-miRNA oligoribonucleotides as indicated. Then GFP-positive cells were sorted and cultured in liquid differentiation media in the absence or presence of activin A. The differentiation state of the cells was determined by Wright-Giemsa staining of cytospin preparations, and cell numbers at different differentiation stages (F) or total cell numbers (G) were counted at the indicated times. For panel F, approximately 300 cells were counted from 3 different fields.