Examination of oncogenic effect of Bcl11a overexpression using the BMTT assay in mice. (A) The BMTT scheme used in this study. (B) Survival analysis on the BMTT-recipient mice, which received Bcl11a- or empty vector (used as a negative control)–transduced Nf1-intact (“Nf1^(+/+).EV” and “Nf1^(+/+). Bcl11a”) or Nf1-deficient bone marrow cells (“Nf1^(Flox/Fcr).EV” and “Nf1^(Flox/Fcr).Bcl11a”). (C) Histopathologic examination of the Bcl11a-induced leukemia in the recipient mice. Hematoxylin and eosin staining. Representative images with magnification of 60-fold are shown here. Bars represent 25 μm. Slides were viewed with a Nikon Eclipse E600 bright light microscope (Nikon, Tokyo, Japan) using a Nikon Plan Fluor 40×/0.75 objective lens. Images were acquired using a Spot Insight digital camera (model 14.2, Color Mosaic; Diagnostic Instruments, Sterling Heights, MI), and were processed with Spotsoftware Advanced (version 4.6 for Windows; Diagnostic Instruments) and Adobe Photoshop version 7.0 software (Adobe Systems, San Jose, CA). (D) Determination of donor origin of the Bcl11a-induced leukemia developing in the BMTT-recipient mice. Antibodies staining for CD45.1 or CD45.2 cell-surface molecules were used. (E,F) Determination of cell-lineage origin of the Bcl11a leukemia developed in the BMTT-recipient mice. Antibodies against Mac-1, Gr-1, B220, or TCR-β were used to stain lymph nodes and bone marrows harvested from either healthy C57BL/6J mice or mice transplanted with Bcl11a-transduced Nf1-deficient bone marrow cells to collect evidence for acute myeloid leukemia (E) or T-cell acute lymphoid leukemia (F).