Fosb and Mn1 can contribute to AML maintenance or development of AML with MLL-AF9. (A) Growth curve of U937 cells with and without induction of shRNA against FOSB with doxycycline. The x-axis represents days after plating cells at 0.5 million cells/well; the y-axis is the number of cells per well in millions of cells. Doxycycline was added 24 hours after plating. Error bars reflect the SD of 3 wells/condition/cell line. Cell lines and conditions were labeled according to the legend (*significant P value < .05; **significant P value < .01). P values reflect paired t test comparing E3 or F10 shRNA U937 cells to U937 cells with a scrambled control, all treated with doxycycline. (B) shRNA competitive proliferation assay in Tet-On MLL-AF9;Nras^G12D AMLs (expressing rtTA3) transduced with indicated TRMPV-Neo-shRNA targeting Fosb or Renilla luciferase. Cells were drug selected and mixed with 20%-40% untransduced cells, followed by shRNA induction with doxycycline. The percentage of shRNA-expressing cells is monitored over time. Error bars represent 2 or 3 independent experiments except in the case of 436 (*significant P value < .05). (C) Kaplan-Meier survival curve of mice injected with BM transduced with a retrovirus containing MN1 or GFP. The x-axis is the number of days after injection of cells into irradiated recipient mice; the y-axis is the percentage of survival. The study was ended at 150 days. Survival of mice receiving MN1-transduced Mll-AF9 BM was significantly shorter than that of mice with MN1-transduced WT BM (P = .0046) and mice with GFP-transduced Mll-AF9 BM (P < .0001); *significant P value < .001, **significant P value < .0001. A total of 52 mice were used in the study: Mll-AF9 BM/MN1 (n = 20), WT BM/MN1 (n = 6), Mll-AF9 BM/GFP (n = 21), and WT BM/GFP (n = 5).