Detection of differentiation-related antigens, PML-RARα, and HIF-1α proteins. (A-D) When leukemic mice in normal oxygen were moribund in the same experiments as those described in Figure 2, all mice were killed and cells from BM and spleen were harvested. (A) Myeloid cells in R1 or R2 region as shown in Figure 3A were analyzed for Gr-1 and Mac-1 expression on flow cytometry. Each column represent the mean percentages of Gr-1⁺ and Mac-1⁺ cells of 3 or 4 mice in an independent experiment; error bars represent SD. Compared with leukemic mice in normal oxygen (iii): *P = 6.746 × 10^–5, **P = 5.303 × 10^–5 for Gr-1⁺ cells; #P = .002, ##P = .014 for Mac-1⁺ cells in BM; &P = .023, &&P = .024 for Gr-1⁺ cells; and P = .002 for Mac-1⁺ cells in spleen cells. (B-C) Isolated cells were collected onto slides by cytospin and stained, respectively, with anti–mouse Gr-1 (B) and anti–human PML antibodies (C) as described in “Materials and methods.” Immunofluorescence was observed under a microscope (original magnification × 100/1.30 NA oil objective lens). Red staining beside cytoplasms (B) and green staining in the nuclei (C) represent Gr-1 and PML, respectively. (D) Splenocytes from the indicated mice were extracted and PML-RARα proteins were detected by Western blot with NB4 cells as positive control. Symbols i to v represent mice with the same treatment as in Figure 2. (E-F) On the first day after transplantation of leukemic cells, mice were housed into intermittent hypoxia (E), or on the seventh day after transplantation of leukemic cells, mice were treated with 15 μg/g body wt of CoCl₂ or 50 μg/g body wt of DFO (F). HIF-1α protein of liver extracts was dynamically detected by Western blots. For panels D-F, equal protein loading was confirmed by Ponceau S staining.