Lmo2 impedes thymocyte maturation at the DN2 stage. (A) Bone marrow cells from Arf^+/+ and Arf^−/− animals were transduced with Lmo2-mCherry and transplanted into lethally irradiated recipient animals. At 91 days after transplantation, mice were killed and thymocytes were analyzed by fluorescence-activated flow cytometry for mature (CD4 and CD8) and immature (CD44 and CD25) T-cell markers. The control was a normal age-matched thymus stained for the same markers. (B) Antibody-dependent microbead-mediated depletion was used to remove the double-positive (CD4⁺CD8⁺) and single-positive (CD4⁺ and CD8⁺) cells from the thymus of 3 Arf^+/+ and 3 Arf^−/− animals. The remaining double-negative thymocytes were transduced with either MSCV-IRES-mCherry or MSCV-Lmo2-IRES-mCherry. Transduced thymocytes were cultured in vitro on OP9-DL1 stromal cells together with IL-7 and FLT-3 ligand. After 14 days, transduced thymocytes expressing mCherry were immunostained as in panel A, sorted to obtain the DN2 population (CD44⁺CD25⁺), and replated at the same density on OP9-DL1 stromal cells. Seven days later, cells were reanalyzed by flow cytometry for the same markers. (C) The absolute number of cells in Lmo2, Arf^+/+ and Lmo2, Arf^−/− populations at day 4, 8, and 11 after the sort were counted. Consistent results were obtained in 2 replicate experiments.