Figure 6
Figure 6. Phenotypic defects in the HSC compartment of Mll5−/− mice are associated with reduced reconstitution potential and increased cycling of CD34− LSK cells. (A) Inefficient rescue of lethally irradiated mice with Mll5−/− BM grafts. C57BL/6 females were lethally irradiated and grafted with FACS-sorted Lin-negative BM cells from either Mll5−/− mice (KO/KO) or WT littermates (WT/WT). Results are a composite of 2 independent experiments (P < .001; log rank test). (B) Competitive reconstitution. Lin-negative donor (CD45.2) BM cells were mixed with an equal number of Lin-negative CD45.1 competitor cells and injected into lethally irradiated CD45.1 mice. Peripheral blood was analyzed 8 (left) and 16 weeks (right) after reconstitution by FACS for donor-derived B lymphoid (CD19), T lymphoid (CD3), and myeloid (Gr1+/Mac1+) cells. Individual recipients reconstituted with Mll5−/− donor cells (○) or donor cells from WT littermates (●) are shown (data from 2 independent experiments combined). (C,D) Cell cycle analysis of LSK cells by in vivo BrdU labeling. (C) Increased cycling of CD34-negative LSK cells in the absence of Mll5 as assessed by 7-amino-actinomycin D/BrdU staining and FACS analysis 2 hours after intraperitoneal injection of BrdU into Mll5-deficient (KO/KO) mice and WT (WT/WT) littermate controls. (Left panels) Representative dot plots from 1 of 4 independent experiments. (Right bar graphs) Average percentage of BrdU-labeled cells in the CD34-negative (top) and CD34-positive (bottom) LSK compartment (**P < .05; n.s. indicates not significant; n = 4). (D) Reduced frequency of noncycling LSK cells in the absence of Mll5. BrdU was given orally to Mll5−/− (KO/KO) mice and WT littermate controls (WT/WT) for 7 days. BrdU incorporation in CD34− (top) and CD34+ LSK cells was evaluated by FACS analysis. (Left panels) Representative dot plots from 1 of 4 independent experiments. (Right bar graphs) Average percentage of noncycling (BrdU-negative) cells in the CD34-negative (top) and CD34-positive (bottom) LSK compartment (**P < .05, *P = .12; n = 4).

Phenotypic defects in the HSC compartment of Mll5−/− mice are associated with reduced reconstitution potential and increased cycling of CD34− LSK cells. (A) Inefficient rescue of lethally irradiated mice with Mll5−/− BM grafts. C57BL/6 females were lethally irradiated and grafted with FACS-sorted Lin-negative BM cells from either Mll5−/− mice (KO/KO) or WT littermates (WT/WT). Results are a composite of 2 independent experiments (P < .001; log rank test). (B) Competitive reconstitution. Lin-negative donor (CD45.2) BM cells were mixed with an equal number of Lin-negative CD45.1 competitor cells and injected into lethally irradiated CD45.1 mice. Peripheral blood was analyzed 8 (left) and 16 weeks (right) after reconstitution by FACS for donor-derived B lymphoid (CD19), T lymphoid (CD3), and myeloid (Gr1+/Mac1+) cells. Individual recipients reconstituted with Mll5−/− donor cells (○) or donor cells from WT littermates (●) are shown (data from 2 independent experiments combined). (C,D) Cell cycle analysis of LSK cells by in vivo BrdU labeling. (C) Increased cycling of CD34-negative LSK cells in the absence of Mll5 as assessed by 7-amino-actinomycin D/BrdU staining and FACS analysis 2 hours after intraperitoneal injection of BrdU into Mll5-deficient (KO/KO) mice and WT (WT/WT) littermate controls. (Left panels) Representative dot plots from 1 of 4 independent experiments. (Right bar graphs) Average percentage of BrdU-labeled cells in the CD34-negative (top) and CD34-positive (bottom) LSK compartment (**P < .05; n.s. indicates not significant; n = 4). (D) Reduced frequency of noncycling LSK cells in the absence of Mll5. BrdU was given orally to Mll5−/− (KO/KO) mice and WT littermate controls (WT/WT) for 7 days. BrdU incorporation in CD34− (top) and CD34+ LSK cells was evaluated by FACS analysis. (Left panels) Representative dot plots from 1 of 4 independent experiments. (Right bar graphs) Average percentage of noncycling (BrdU-negative) cells in the CD34-negative (top) and CD34-positive (bottom) LSK compartment (**P < .05, *P = .12; n = 4).

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