miR-125b cooperates with Gata1s to induce leukemia in vivo. (A) In vitro and in vivo setup for modeling synergy between Gata1s and the members of the miR-99a-125b tricistron (left). Percentage of Gata1s FLCs transduced with different miRNA permutations (marked by dTomato [miR-125b], mTagBFP2 [let-7c], and GFP [miR-99a]) normalized to that on day 0 (n = 4; 1-way ANOVA) (right). (B) Bar graph showing the percentage of stem/progenitor cells (CD117⁺), mature megakaryocytes (MK, CD41⁺CD42d⁺), and erythroid cells (ERY, CD71⁺Ter-119⁺) after 6 days of differentiation. Cells shown are Gata1s FLCs transduced with miR-125b or miR-ctrl (n = 3, paired Student t test). (C) Representative methylcellulose-based CFU assays from 1 of 4 independent experiments. Gata1s FLCs transduced with miR-ctrl or miR-125b are depicted in complete or low (Thpo 20 ng/mL) cytokine conditions. (D) The number of megakaryocyte colonies after serial replating of miR-125b Gata1s FLCs in methylcellulose-based medium with low (Thpo 20 ng/mL) cytokine conditions, compared with miR-ctrl Gata1s FLCs (n = 4, unpaired Student t test). (E) Classification of colonies after plating miR-125b or miR-ctrl Gata1s FLCs in methylcellulose-based CFU assays under complete cytokine conditions (n = 2, 2-way ANOVA). Combined granulocytic (CFU-G) and monocytic (CFU-M) CFUs: granulocytic/monocytic (CFU-G/M); and CFU/BFU-E, erythroid. (F-H) Analysis of mouse recipients of Gata1s FLCs overexpressing miR-125b or miR-ctrl (n = 10 per group), including comparisons of Kaplan-Meier survival curves (log-rank test) (F), spleen weights (unpaired Student t test) (G), and representative flow cytometry plots of bone marrow–derived leukemic cells (H) in the diseased mice. miR-125b–overexpressing Gata1s FLC-derived blasts are highlighted in red. The average percentage of miR-125b⁺ blasts belonging to each immunophenotype is indicated in each corresponding gate. Data are the mean ± standard deviation. FP⁺, fluorescent protein positive; n.s., not significant.