Hematopoiesis-specific deletion of Hif-1α does not affect HSC survival and maintenance and their ability to respond to 5-FU–induced stress. (A) Experimental design. A total of 500 000 donor-derived (CD45.2⁺) unfractionated BM cells from untreated Hif-1α^fl/fl;Mx1-Cre and control (without Mx1-Cre) mice were transplanted into lethally irradiated syngeneic CD45.1⁺/CD45.2⁺ recipient mice (together with 500 000 CD45.1⁺ competitor BM cells). Two independent donors were used per genotype. Eight weeks posttransplantation, the mice received 6 sequential doses of pIpC over a period of 10 days (every alternate day) and were analyzed 2 weeks after last dose of pIpC. CD45.2⁺ LSK cells from the primary recipients were serially transplanted into secondary and tertiary recipients. (B) Percentage of CD45.2⁺ donor-derived cells 8 weeks posttransplantation in the PB of primary recipient mice before pIpC treatment (n = 5-6 per group). (C) Representative gel showing PCR amplification of genomic DNA from donor-derived CD45.2⁺ fraction of the PB of pIpC-treated recipient mice 2 weeks after last pIpC injection. Δ, excised allele; fl, undeleted conditional allele. For PCR controls, we used genomic DNA from c-Kit⁺ cells from BM of Hif-1α^+/+, Hif-1α^fl/fl, and Hif-1α^fl/fl;Vav-iCre mice. (D) Percentage of CD45.2⁺ donor-derived cells in PB 2 weeks after the last pIpC injection (n = 5-6 per group). (E) Percentage of CD45.2⁺ cells in the unfractionated BM (total BM cells) and myeloid (CD11b⁺Gr1⁺), B lymphoid (CD19⁺B220⁺), and erythroid (Ter119⁺) cell compartments of the primary recipient mice. Data are mean ± standard error of the mean (SEM; n = 5-6 per group). (F) Percentage of CD45.2⁺ donor-derived cells measured in BM LK and LSK cell compartments of the primary recipients. Data are mean ± SEM (n = 5-6 per group). (G) Percentage of CD45.2⁺ donor-derived cells in LSK CD48⁻CD150⁺ HSC, LSK CD48⁻CD150⁻ MPP, and primitive progenitor (LSK CD48⁺CD150⁻ HPC-1 and LSK CD48⁺CD150⁺ HPC-2) cell compartments of primary recipients. Data are mean ± SEM (n = 5-6 per group). (H) PB chimerism in secondary recipients of control and Hif-1α^∆/∆ LSK cells sorted from BM of the primary recipients. Data are mean ± SEM (n = 6 per group). (I) Representative gel showing efficient deletion of the conditional alleles of Hif-1α in the CD45.2⁺ BM cells of secondary recipients 16 weeks after transplantation. For PCR controls, we used genomic DNA from c-Kit⁺ cells from BM of Hif-1α^+/+, Hif-1α^fl/fl, and Hif-1α^fl/fl;Vav-iCre mice. (J) Percentage of CD45.2⁺ cells in total BM, myeloid, B lymphoid, and erythroid cell compartments of the secondary recipient mice 16 weeks posttransplantation. Data are mean ± SEM (n = 6 per group). (K-L) Percentage of CD45.2⁺ cells in BM stem and progenitor cell compartments of the secondary recipient mice 16 weeks posttransplantation. Data are mean ± SEM (n = 6 per group). (M) Results of the tertiary transplantation assay. The graph shows the percentage of tertiary recipients with long-term multilineage reconstitution (>0.5% of donor-derived myeloid and lymphoid cells in BM) 16 weeks after tertiary transplantation of Hif-1α^∆/∆ and control LSK cells sorted from BM of the secondary recipients. Data are mean ± SEM (n = 12-19 per group). (N) Percentage of CD45.2⁺ cells in the BM hematopoietic compartments of tertiary recipient mice 16 weeks after transplantation (n = 12-19 per group). (O) Representative gel showing efficient deletion of the conditional alleles of Hif-1α in the CD45.2⁺ BM cells of tertiary recipients 16 weeks after transplantation. For PCR controls, we used genomic DNA from c-Kit⁺ cells from BM of Hif-1α^+/+, Hif-1α^fl/fl, and Hif-1α^fl/fl;Vav-iCre mice. (P) Experimental design. Hif-1α^fl/fl;Vav-iCre and control mice received 3 sequential doses of 5-FU (150 mg/kg; 10 days apart) and were analyzed 10 days after the last 5-FU administration. In parallel, untreated Hif-1α^fl/fl;Vav-iCre and control mice that did not receive 5-FU were also analyzed. (Q) Kaplan-Meier survival curve of Hif-1α^fl/fl;Vav-iCre and control mice treated with 5-FU (ie, +5-FU) (n = 7-8 per group) or those that were not treated with 5-FU (ie, −5-FU) (n = 4-6 per group). Arrows in the graph indicate 5-FU administration. (R) Total numbers (per 2 femurs and 2 tibias) of BM white blood cells, LSK cells, HSCs, and LK cells from the mice described in panels P and Q. Data are mean ± SEM (n = 4-6 per group). At least 2 independent experiments were performed for all analyses.