Figure 6. ASXL1 truncations synergize with BAP1 and TET2 loss-of-function to skew commitment to the myeloid lineage in vivo.

(a) Lineage-negative, c-Kit+ (LK) cells isolated from mouse bone marrow and transduced with either MiG-empty or ASXL1(1–479)+BAP1. Transduced cells were expanded in media supplemented with 10 ng ml⁻¹ IL-3, fluorescence-activated cell sorted (FACS) for Thy1.1 and green fluorescent protein (GFP) expression on day 5, and expanded in liquid medium supplemented with 10 ng ml⁻¹ IL-3 till day 7. On day 7, the cells were washed extensively to remove traces of IL-3. Cells (10⁵) were plated in triplicate in 35-mm dishes in methylcellulose medium supplemented with 10 ng ml⁻¹ GM-CSF. Numbers of CFU-GM were counted on day 14. Statistical significance was determined by two-tailed t-test. Representative image and FACS profile of a CFU-GM are shown to the right. (b) 5-FU-treated bone marrow cells were harvested from WT and TET2-deficient donor mice as described in Methods. The donor cells were transduced as indicated 24 h post transduction; 1.2 × 10⁶ cells were transplanted into irradiated recipient mice by intravenous injection. (c) The bone marrow was harvested from recipient mice 6 months post transfer. Transduced cells were identified based on expression of GFP and Thy1.1 reporters. Shown are percentages of BAP1, ASXL1(1–479) and doubly transduced cells in the bone marrows of recipient mice 6 months post transfer. Each dot represents one mouse. (d) Percentages of CD11b (myeloid)- and B220 (B-lymphoid)-positive cells that were transduced with MiG-empty, BAP1 alone or ASXL1(1–479)+BAP1 retroviruses were determined by flow cytometry. Data shown are from recipients that received cells from TET2-deficient donors; percentages represent mean±s.d. from five recipients. Statistical significance was determined by two-tailed t-test; n=5. (e) Representative FACS plots of CD11b- and B220-positive cells in the bone marrow of recipient mice that were transplanted with Tet2^−/− bone marrow cells 6 months post transfer.