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Nupr1 regulates the quiescence threshold of HSC
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Figure 6. Reversion of p53 expression by allelic depletion of the Mdm2 gene offsets the repopulating advantage of Nupr1-/- hematopoietic stem cells. (A) Representative plots of hematopoietic stem cell (HSC) analysis by flow cytometry from wild-type (WT), Nupr1-/-, Nupr1-/-Mdm2+/- and Mdm2+/- mice bone marrow. (B) Statistical analysis of WT, Nupr1-/-, Nupr1-/-Mdm2+/- and Mdm2+/- HSC number. Data were analyzed by two-way analysis of variance (ANOVA). n=5. BMNC: bone marrow nucleated cells; n.s.: not significant. (C) Donor bone marrow cells (2.5×105) from WT (black) Nupr1-/- (red), Nupr1-/-Mdm2+/- (blue) (CD45.2) or Mdm2+/- (purple) mice were transplanted into lethally irradiated recipient mice (CD45.1) along with 2.5×105 recipient bone marrow cells. Data were analyzed using an unpaired Student t- test and are represented as mean ± standard deviation (SD). WT, n=5; Nupr1-/-, n=5 mice; Nupr1-/-Mdm2+/-, n=6 mice; Mdm2+/-: n=5. *P<0.05, ***P<0.001. PB: peripheral blood. (D) Flow cytometry analysis of donor-derived HSC and recipient HSC in bone marrow of recipient mice 4 months after transplantation. HSC were gated as Lin– (i.e., CD2–, CD3–, CD4–, CD8–, B220–, Gr1–, CD11b–, Ter119–) CD48– Sca1+ c-Kit+ CD150+ CD34– CD135– cells. Plots from one representative mouse of each group are shown. (E) Statistical analysis of the percentage and absolute number of donor-derived HSC in recipient mice 4 months after transplantation. Data were analyzed by one-way ANOVA and are represented as mean ± SD. WT, n=5; Nupr1-/-, n=5 mice; Nupr1-/-Mdm2+/-, n=6 mice; Mdm2+/-: n=5. ***P<0.001.
mice (median value: 14.13%, P=0.07) (Figure 7A-C). The competitive transplantation result showed that donor Nupr1fl/flMx1-cre cells were advantaged over WT competi- tors in the peripheral blood of recipients (60%-80%) (Figure 7D). To further investigate whether Nupr1fl/flMx1-cre HSC
dominantly occupy recipient bone marrow, we sacrificed the recipients and analyzed the HSC 16 weeks after trans- plantation. The proportion and absolute number of Nupr1fl/flMx1-cre HSC were significantly greater (~2-fold) than the control HSC competitors in primary recipients
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