Enforced expression of miR-125b modulates granulocytic differentiation of murine Lin− cells in vitro and promotes myeloid cell development in vivo. (A) Colony formation by 1 × 105 Lin−/GFP−, Lin−/miR-ctrl and Lin−/miR-125b cells in methylcellulose cultures supplemented with G-CSF. (B) Microscopic images of colonies (top panel) and morphology of cells isolated from corresponding clonogenic assays (bottom panel); 4× and 60× magnifications of a representative field are shown, respectively. Analysis of (C) BM and (D) spleen cells of 10-week BM chimeras. The top panels show representative density plots of donor-derived cells stained for GR-1, CD11b, and CD11c and electronically gated on GFP− (left) and GFP+ (right) cells. In the bottom panels, lines indicate mean percentage of GFP− and GFP+ cells according to gates indicated in the corresponding top panels. Each dot represents an individual mouse. Data are pooled from 2 independent experiments. (E) Colony formation by Lin− cells isolated from BM chimeras described in panels C and D. 1 × 103 Lin−/miR-ctrl or Lin−/miR-125b cells (1 × 103) were cultured in methylcellulose containing SCF, IL-3, IL-6, and Epo. (F) Microscopic images of colonies from the experiment described in panel E; a 60× magnification of the representative field is shown. Mean of 4 (panel A), 2 (panels C,D,E) and representative experiments are shown, respectively. *P < .05 compared with Lin−/miR-ctrl cells.
