Cux1^low cells aberrantly proliferate and expand following DNA-damaging alkylating agents. (A) Bone marrow from Ren, Cux1^mid, and Cux1^low mice was transplanted at a 1:4 ratio with competitor bone marrow, and recipients were treated continuously with Dox beginning the day of transplant. Two doses of ENU (100 mg/kg) were administered 9 days apart, starting 5 weeks after transplant. (B) The contributions of Ren (n = 9), Cux1^mid (n = 8), and Cux1^low (n = 9) cells to the peripheral blood were monitored by flow cytometry. The ratio of Ren, Cux1^mid, or Cux1^low cells (CD45.2) to competitor cells (CD45.1) is shown. The mean ± SEM is shown. A mixed-effects analysis with the Geisser-Greenhouse correction is shown. Two independent biological replicates were performed (n = 4-5 mice per replicate per genotype). (C-D) At 16 weeks, the mice were euthanized and the contributions of Ren, Cux1^mid, and Cux1^low (CD45.2) to the hematopoietic stem and progenitor populations were measured by flow cytometry in the bone marrow (C) and spleen (D). LSK cells and myeloid progenitors (Lin⁻/Sca1⁻/c-Kit⁺) are shown (left). LSK cells were further gated for long-term HSCs (LT-HSC; CD150⁺/CD48⁻), short-term HSCs (ST-HSC; CD48⁻/CD150⁻), and multipotent progenitors (MPP; CD48⁺/CD150⁻) (center). Within the progenitor population, cells were separated into common myeloid progenitors (CMP; CD34⁺/CD16/32^low), granulocyte-monocyte progenitor (GMP; CD34⁺/CD16/32^high), and megakaryocyte-erythroid progenitors (MEP; CD34⁻/CD16/32⁻) (right). The mean ± SD and P values from a 1-way ANOVA comparison are shown. Two independent biological replicates were performed. (E) Proliferation was measured in Ren and Cux1^low mice after ENU administration. Mice were given 2 doses of ENU, 9 days apart. Six hours after the second dose of ENU, BrdU was administered. BrdU⁺ cells were measured by flow cytometry 12 hours after BrdU injection in the bone marrow HPSC population (Lin⁻). Representative flow plots are shown. The mean ± SD and Student t test P value is shown. (F) Apoptosis was measured in the same cells by flow cytometry using antibodies for cleaved poly (ADP-ribose) polymerase (cPARP). Three independent biological replicates were performed. SSC-A, side scatter area; WT, wild type.