HIF1α is required for survival maintenance of LSCs. (A) The cell-cycle analysis of normal LSK cells and BCR-ABL–expressing LSK cells. The percentages of HIF1α^−/− BCR-ABL–expressing LSK cells were low in the S phase and higher in the G₀-G₁ phase, compared with WT BCR-ABL–expressing LSK cells (n = 6). However, normal WT and HIF1α^−/− LSK cells showed similar percentages of cells in G₀-G₁, S, and G₂-M phases. (B) The cell-cycle analysis of BCR-ABL–expressing LSK cells using Ki67 and Hoechst 33 342 showed an accumulation of G₁ phase of HIF1α^−/− BCR-ABL–expressing LSK cells. (C) RT-PCR analysis showed elevated expression of cell-cycle inhibitors p16^Ink4a, p19^Arf, and p57 in the sorted LSK cells and BCR-ABL–expressing LSK cells from HIF1α^−/− control and CML mice compared with the sorted LSK cells and BCR-ABL–expressing LSK cells from WT control and CML mice. BM cells from control and CML mice at day 14 post-BMT were collected, and LSK cells and BCR-ABL–expressing LSK cells were sorted for extracting total RNA. Mean values (± SD) are shown; **P < .01; ***P < .001. (D) The apoptosis of normal LSK cells and BCR-ABL–expressing LSK cells from BM of control and CML mice (n = 6). HIF1α deletion induced the apoptosis of BCR-ABL–expressing LSK cells, but did not affect the apoptosis of normal LSK cells. (E) FACS analysis showed a higher percentage of active caspase positive HIF1α^−/− leukemia progenitors. (F) RT-PCR analysis showed elevated expression of p53 in the sorted BCR-ABL–expressing LSK cells from HIF1α^−/− CML mice comparing to the sorted BCR-ABL–expressing LSK cells from control CML mice. Mean values (± SD) are shown; **P < .01. (G) Simultaneous knockdown of p16^Ink4a and p19^Arf rescued the colony-forming ability of HIF1α^−/− BCR-ABL–expressing LSK cells. Western blot analysis showed that protein levels of p16^Ink4a and p19^Arf in p53^−/− MEF and CML leukemia cells were dramatically reduced by shRNA no. 1 that targets both p16^Ink4a and p19^Arf. BM cells from WT and HIF1α^−/− normal or CML mice were infected by shRNA no. 1, and plated into methycellulose media after selection by puromycin. Colonies were counted and cells from the colonies were serially replated. Mean values (± SD) are shown; **P < .01; ***P < .001. (H) Loss of HIF1α did not affect the senescence of normal LSK cells and BCR-ABL–expressing LSK cells. Sorted GFP⁺Lin⁻Sca-1⁺c-Kit⁺ cells from the BM of WT or HIF1α^−/− control and CML mice were stained by X-Gal, and the number of β-galactosidase–expressing cells were counted.