miR-125b accelerates leukemogenesis induced by MLL-AF9. (A) Schematics of the experiment, in which GMPs from Ri125b mice were transduced with MLL-AF9-ΔNGFR (AF9) and injected into wild-type recipients. Recipients were randomly assigned to the Dox treatment group or the −Dox group. (B) Representative flow cytometry plots demonstrating the detection of GFP⁺ΔNGFR⁺ cells in peripheral blood 4 weeks after transplantation in +Dox and −Dox mice. GFP marks miR-125b induction and ΔNGFR marks MLL-AF9–transduced cells. (C) Kaplan-Meier survival curve of primary AF9+Ri125b+Dox and AF9+Ri125b−Dox cohorts (P < .001). (D) Representative Giemsa staining of bone marrow cells from MLL-AF9–only or AF9+Ri125b+Dox leukemia mice, showing similar blast morphology. Images were captured by an Olympus BX51 (Olympus) using a SensiCam QE camera (PCO) and IPLab 4.0.8 software (BioVision Technologies). (E) Left: bone marrow blast sizes of MLL-AF9–only and AF9+Ri125b+Dox leukemia (n = 40 cells). Right: bone marrow blast percentages quantified from AF9+Ri125b+Dox and AF9+Ri125b−Dox leukemia-bearing mice (n = 3). (F) Schematics of experiment to analyze secondary leukemogenesis for dependence on miR-125b overexpression. Primary AML cells from AF9+Ri125b+Dox mice were transplanted into lethally irradiated wild-type mice. Secondary recipients were randomized into cohorts for continuation or withdrawal of Dox. (G) Kaplan-Meier survival curves for AF9+Ri125b secondary AML cohorts with continued Dox and Dox withdrawal, with results from 3 primary AML donors. NS, not significant.