Impact of hypoxia and HIF-1α on BM cell differentiation in vitro. (A-E) BM single-cell suspensions of either WT or HIF-1α^fl/fl LysM-Cre (HIF-1-KO) mice were generated. A total of 2 × 10⁶ cells/mL were seeded in Ultra-Low attachment plates. (A-D) Cells were cultured with 200 ng/mL Flt3-L at 20% or 5% O₂ for 9 days. (A,E,H) Total numbers of the indicated cell populations were determined by flow cytometry. For gating strategies, see supplemental Methods and supplemental Figure 2. (A) Data are mean ± SEM of cells cultured from 6 animals of each genotype (3 independent experiments using cells of 2 mice each). (B) Relative changes in cell counts of the indicated populations under 5% versus 20% O₂ in WT or HIF-1-KO cultures were compared. The red dotted line indicates the 20% O₂ control values. Data are mean ± SEM of cells cultured from 10 animals of each genotype (4 independent experiments using cells of 2 or 3 mice each). (C) Relative mRNA expression of E2-2, ID2, and PU.1 quantified by quantitative PCR is shown. mRNA levels of 20% O₂ WT or HIF-1-KO were set to 1. Data are mean ± SEM of 4 independent experiments using pooled cells of 2 or 3 mice each. (D) IFN-α secreted from 10⁴ cells in 10 μg/mL CpG-stimulated BM cultures was determined by ELISA. Data are mean ± SEM of cells cultured from 6 animals of each genotype (3 independent experiments using cells of 2 mice each). (E) Cells were cultured with 200 ng/mL Flt3-L with or without 100μM DMOG for 9 days. Data are mean ± SEM of cells cultured from 8 animals of each genotype (3 independent experiments using cells of 2 or 3 mice each). (F-G) Monocyte/DC progenitors and common DC progenitors were isolated from whole BM cell suspensions of WT or HIF-1-KO mice using untouched magnetic separation followed by FACS sorting (see supplemental Methods and supplemental Figure 4), and 10⁴ cells/well were seeded in Ultra-Low attachment plates. Cells were cultured with Flt3-L at 20% or 5% O₂ for 48 hours. (F) Intracellular expression of HIF-1α and ID2 in cultured MDPs/CDPs was quantified by flow cytometry using biotin-coupled HIF-1α and ID2 antibodies and streptavidin-PE-CF495 (see supplemental Methods and supplemental Figure 5). A representative histogram of 3 independent experiments using pooled cells of 2 or 3 mice each is shown. (G) The number of CD11c-expressing cells generated from MDPs/CDPs on 48-hour culture is shown. Data are mean ± SEM of 3 independent experiments using pooled cells of 2 or 3 mice each. (H) A total of 2 × 10⁶ BM cells of ID2-KO and respective WT mice were cultured with 200 ng/mL Flt3-L at 20% or 5% O₂ for 9 days. Data are mean ± SEM of cells cultured from 8 animals of each genotype (3 independent experiments using cells of 2 or 3 mice each). Data were analyzed using GraphPad Prism Version 5.0 for Windows. P values were calculated using Student t test (B) or 1-way ANOVA (A,C-E,G-H) with Bonferroni correction. Significant differences between experimental groups: *P < .05, **P < .01, ***P < .001.