Knockdown expression of Pbx3 by shRNA inhibits cell transformation mediated by MLL-AF9. Colony-forming and replating assays of mouse normal BM progenitor cells transduced with MSCVneo-MLL-AF9⁺pGFP-V-RS-scrambled (ie, MA9) or MSCVneo-MLL-AF9⁺ pGFP-V-RS-Pbx3 shRNA (ie, MA9⁺shPbx3). Duplicates were plated for each combination with 1 × 10⁴ cells per dish, and every 7 days the cells were replated for up to 3 passages. Mean values and standard deviations (mean ± SD) from 2 independent experiments are shown (*P < .01; **P < .001, 2-tailed t test). (A) Numbers of colonies per dish (only colonies with ≥ 50 cells/colony were counted; 1 × 10⁴ input cells) in each passage are shown. (B) Average numbers of colony cells per dish are shown. (C) Morphology of colonies of secondary passage. Scale bars represent 100 μm. (D) Morphology of cells of secondary passage. Cells were stained with Wright-Giemsa. Scale bars represent 10 μm. (E) Flow cytometric analysis of colony-forming cells (secondary passage) with APC-labeled anti-CD117 (ie, c-Kit) antibody and eFluor 450–labeled anti-CD11b (ie, Mac-1) antibody. (F) qPCR assay of the endogenous expression of Pbx1, Pbx2, Pbx3, Mef2c, Myb, and Flt3 in colony cells collected from the secondary passage.