Figure 5
Figure 5. THPO-independent role of FL in regulation of LMPPs but not HSCs. (A) Number of LSKFLT3−, LSKFLT3+, and LMPPs (LSKFLT3hi) in WT, Fl−/−, Thpo −/−, and Fl−/− × Thpo −/− per tibia. Each dot represents an individual mouse. Data are from 3 to 4 experiments. Statistical significance was tested between WT and Fl−/−; Fl−/− and Fl−/− × Thpo−/− (it was not tested in LSKFlt3−); and Thpo−/− and Fl−/− × Thpo−/−. Due to multiple comparisons, Bonferroni-Holms correction of P values was performed (see “Statistics”). ns indicates not significant; **P < .01, ***P < .001. (B) Competitive transplantation assay: 1 million donor BM cells (CD45.1) of indicated genotypes were transplanted together with 1 million WT BM competitor (CD45.2) cells. Representative FACS profile from reconstitution analysis of peripheral blood from the different genotypes at 4 months posttransplantation. Cells were first gated as negative for NK1.1, further gating to identify the myeloid lineage and relative contribution of CD45.1 and CD45.2 is indicated by the gates and arrows. (C) Competitive transplantation assay: peripheral blood analysis at 4 months posttransplantation. Gating strategy is shown in panel B. (Top left panel) The percentage of CD45.1+ cells of total CD45 (CD45.1 and CD45.2) cells; (top right panel) the percentage of CD45.1+ myeloid cells (of total myeloid cells). (Bottom left panel) The percentage of CD45.1+ T cells (of total T cells); (bottom right panel) the percentage of CD45.1+ B cells (of total B cells). Myeloid cells were defined as NK1.1−Mac-1+, B cells as CD19+ and T cells as CD4/CD8+. Each dot represents an individual mouse. Numbers indicate frequencies of reconstituted mice. Mice were defined as reconstituted when at least 0.1% donor (CD45.1) cells were detected among total CD45+ cells, and lineage (myeloid, T, and B cell) reconstituted if a minimum of 0.02% of all CD45+ cells were CD45.1+ and coexpressing the markers for the specific lineage investigated. Data are from 2 experiments with 6 to 7 mice analyzed per genotype and experiment.

THPO-independent role of FL in regulation of LMPPs but not HSCs. (A) Number of LSKFLT3−, LSKFLT3+, and LMPPs (LSKFLT3hi) in WT, Fl−/−, Thpo−/−, and Fl−/− × Thpo−/− per tibia. Each dot represents an individual mouse. Data are from 3 to 4 experiments. Statistical significance was tested between WT and Fl−/−; Fl−/− and Fl−/− × Thpo−/− (it was not tested in LSKFlt3−); and Thpo−/− and Fl−/− × Thpo−/−. Due to multiple comparisons, Bonferroni-Holms correction of P values was performed (see “Statistics”). ns indicates not significant; **P < .01, ***P < .001. (B) Competitive transplantation assay: 1 million donor BM cells (CD45.1) of indicated genotypes were transplanted together with 1 million WT BM competitor (CD45.2) cells. Representative FACS profile from reconstitution analysis of peripheral blood from the different genotypes at 4 months posttransplantation. Cells were first gated as negative for NK1.1, further gating to identify the myeloid lineage and relative contribution of CD45.1 and CD45.2 is indicated by the gates and arrows. (C) Competitive transplantation assay: peripheral blood analysis at 4 months posttransplantation. Gating strategy is shown in panel B. (Top left panel) The percentage of CD45.1+ cells of total CD45 (CD45.1 and CD45.2) cells; (top right panel) the percentage of CD45.1+ myeloid cells (of total myeloid cells). (Bottom left panel) The percentage of CD45.1+ T cells (of total T cells); (bottom right panel) the percentage of CD45.1+ B cells (of total B cells). Myeloid cells were defined as NK1.1−Mac-1+, B cells as CD19+ and T cells as CD4/CD8+. Each dot represents an individual mouse. Numbers indicate frequencies of reconstituted mice. Mice were defined as reconstituted when at least 0.1% donor (CD45.1) cells were detected among total CD45+ cells, and lineage (myeloid, T, and B cell) reconstituted if a minimum of 0.02% of all CD45+ cells were CD45.1+ and coexpressing the markers for the specific lineage investigated. Data are from 2 experiments with 6 to 7 mice analyzed per genotype and experiment.

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