Figure 4
Figure 4. Kdm2b/Jhdm1 is required for Hoxa9-Meis1-induced AML development in vivo. (A) Flow chart of experimental procedure for BM transplantation assays. To examine the role of Kdm2b/Jhdm1b in Hoxa9-Meis1-induced AML development in vivo, c-kit+ progenitors were isolated from E14.5 fetal liver (FL) of Ly5.2 C57BL/6 embryos. After lentiviral (LV) transduction with vectors expressing various combinations of proteins and shRNAs, the genetically modified cells were mixed with normal irradiation protector cells and were transplanted into lethally irradiated Ly5.1 C57BL/6 mice. (B) FACS analysis of the accumulation kinetics of the genetically modified donor cells (GFP, CKD-HMG, and J1bKD-HMG) in the peripheral blood of recipients. The results indicate that Kdm2b/Jhdm1b KD cells (J1bKD-HMG) failed to repopulate in the Mac-1+ myeloid lineage, whereas the control KD cells (CKD-HMG) can. (C) The percentage of genetically modified cells, marked by GFP, contribute to the myeloid lineage (Gr-1+ and Mac-1+) of peripheral blood at various times after transplantation. The results show that Kdm2b/Jhdm1b KD inhibits repopulating by Hoxa9-Meis1-transduced cells in recipient mice. The percentage of GFP+ cells in a particular lineage is calculated by dividing GFP and lineage marker double-positive cells with the total lineage marker-positive cells. All error bars represent SD (n = 10). (D) Splenomegaly of mice that received a transplant with lentiviral-transduced BM cells expressing CKD-HMG. Shown is a representative picture of spleens harvested from mice 6 weeks after transplantation of BM cells transduced with lentiviral vectors expressing GFP, J1bKD-HMG, or CKD-HMG. Bar size represents 1.0 cm. (E) H&E staining shows leukemic infiltration of multiple organs (spleen, kidney, and lung) in mice that received a transplant with Hoxa9-Meis1-induced leukemic cells (CKD-HMG), whereas there is no obvious leukemic cell infiltration in mice that received a transplant with BM cells transduced with lentiviral vectors expressing GFP or J1bKD-HMG cells. (F) May-Grünwald/Giemsa staining shows typical leukemic cells in the peripheral blood of mice that received a transplant with CKD-HMG cells. Bar size represents 50 μm. (G) Survival curve shows mice that received a transplant with GFP and J1bKD-HMG cells survived ≥ 70 days after transplantation, whereas the mice that received a transplant with CKD-HMG cells all died within 70 days after transplantation.

Kdm2b/Jhdm1 is required for Hoxa9-Meis1-induced AML development in vivo. (A) Flow chart of experimental procedure for BM transplantation assays. To examine the role of Kdm2b/Jhdm1b in Hoxa9-Meis1-induced AML development in vivo, c-kit+ progenitors were isolated from E14.5 fetal liver (FL) of Ly5.2 C57BL/6 embryos. After lentiviral (LV) transduction with vectors expressing various combinations of proteins and shRNAs, the genetically modified cells were mixed with normal irradiation protector cells and were transplanted into lethally irradiated Ly5.1 C57BL/6 mice. (B) FACS analysis of the accumulation kinetics of the genetically modified donor cells (GFP, CKD-HMG, and J1bKD-HMG) in the peripheral blood of recipients. The results indicate that Kdm2b/Jhdm1b KD cells (J1bKD-HMG) failed to repopulate in the Mac-1+ myeloid lineage, whereas the control KD cells (CKD-HMG) can. (C) The percentage of genetically modified cells, marked by GFP, contribute to the myeloid lineage (Gr-1+ and Mac-1+) of peripheral blood at various times after transplantation. The results show that Kdm2b/Jhdm1b KD inhibits repopulating by Hoxa9-Meis1-transduced cells in recipient mice. The percentage of GFP+ cells in a particular lineage is calculated by dividing GFP and lineage marker double-positive cells with the total lineage marker-positive cells. All error bars represent SD (n = 10). (D) Splenomegaly of mice that received a transplant with lentiviral-transduced BM cells expressing CKD-HMG. Shown is a representative picture of spleens harvested from mice 6 weeks after transplantation of BM cells transduced with lentiviral vectors expressing GFP, J1bKD-HMG, or CKD-HMG. Bar size represents 1.0 cm. (E) H&E staining shows leukemic infiltration of multiple organs (spleen, kidney, and lung) in mice that received a transplant with Hoxa9-Meis1-induced leukemic cells (CKD-HMG), whereas there is no obvious leukemic cell infiltration in mice that received a transplant with BM cells transduced with lentiviral vectors expressing GFP or J1bKD-HMG cells. (F) May-Grünwald/Giemsa staining shows typical leukemic cells in the peripheral blood of mice that received a transplant with CKD-HMG cells. Bar size represents 50 μm. (G) Survival curve shows mice that received a transplant with GFP and J1bKD-HMG cells survived ≥ 70 days after transplantation, whereas the mice that received a transplant with CKD-HMG cells all died within 70 days after transplantation.

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