MiR-24 Is Required for Hematopoietic Differentiation of Mouse Embryonic Stem Cells
Fig 6
BL-CFCs develop from EBs with antagonized miR-24.
RW4 parental cells, miArrest-Scr clones, and miArrest-24 clones were differentiated in liquid culture for A) 2.75d or B) 3.0d. EBs were dissociated into single cell suspensions and plated in methylcellulose for growth of BL-CFCs. 4d later BL-CFCs were counted. 2 independent miArrest-Scr clones were examined for d2.75 and d3 differentiations. 4 miArrest-24 clones for d2.75 and 7 miArrest-24 clones for d3 were examined. For d2.75, RW4 n = 4, miArrest Scr n = 6, and miArrest-24 n = 8. For d3.0, RW4 n = 8, miArrest Scr n = 8, and miArrest-24 n = 16. C) RNA was isolated from d3, d4, and d5 EBs derived from the indicated ESC clones. Expression of the transcription factor Etv2 was examined. The decrease in expression observed in the miArrest-24 clones compared to uninfected and miArrest-SCR infected RW4 cells was significant at d3 (P<0.006), d4 (P<0.002), and d5 (P<0.006) as determined with by unpaired t-test. D) Twenty BL-CFC colonies from individual cultures were isolated and used to generate cDNA. Quantitative-RT-PCR was performed with the indicated gene specific primers. Data was averaged from results obtained from the differentiation of 3 scrambled clones, and 3 mir-24 KD clones. The decreases in Gata1, Runx1, and Scl expression are significant with respective P values of P<0.001, P< 10-4, and P<10-7.