Fig 1: ASXL1-MT/BAP1 complex impairs multilineage differentiation of haematopoietic progenitors except for differentiation towards monocytes. a, b Murine c-Kit+ bone marrow cells were transduced with vector, ASXL1-MT (MT), or ASXL1-MT-K351R (KR) (coexpressing GFP) together with vector or BAP1 (coexpressing puromycin-resistant gene). After the puromycin selection for 48 h, cells were cultured with cytokines to induce mast cell or monocyte/macrophage differentiation. Mast cell maturation was assessed by the ratio of FceIRa and c-Kit double-positive cells (enriched with mature mast cells) after 9 days of culture (a). Monocyte (CD115+F4/80-) and macrophage (CD115+F4/80+) differentiation was assessed after 7 days of culture (b). (n = 3 each) (c) Murine c-Kit+ bone marrow cells were transduced with vector, ASXL1-MT (MT), or ASXL1-MT-K351R (KR) (coexpressing blasticidin-resistant gene) together with vector or BAP1 (coexpressing puromycin-resistant gene). After the selection with puromycin and blasticidin for 3 days, cells were cultured with cytokines to induce differentiation toward both granulocytes and monocytes. Granulocyte (Gr-1+CD11b+) and monocyte (Gr-1-CD11b+) differentiation was assessed by FACS analyses (left) and Wright–Giemsa staining (right, scale bars: 20 µm) after 5 days of culture. (n = 3) (d–f) Human CB CD34+ cells were transduced with vector, ASXL1-MT (MT), or ASXL1-MT-K351R (KR) (coexpressing GFP) together with vector or BAP1 (coexpressing puromycin-resistant gene). After the puromycin selection for 48 h, cells were cultured in myeloid skewing (d), erythroid-skewing (e), and stem cell-maintaining (f) mediums, respectively. Myeloid maturation of CB cells was assessed by the ratio of CD33+CD66b+ cells after 7 days of culture (d). Erythroid maturation of CB cells was assessed by the ratio of CD79+CD235a+ cells after 5 days of culture (e). HSC maintenance of CB cells was assessed by the ratio of CD34-high cells after 7 days of culture (f). (n = 4 each) Data are shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test