Effects of hyperoxic-hypoxic-hyperoxic treatment on expansion of WT and Fancc^–/–BM lin^– cells. (A) Growth of WT and Fancc^–/– BM lin^– cells over 10 days. Cells were incubated in hyperoxia (21% O₂; Con: control) only or first subjected to 2 cycles of hypoxia (1% O₂) for 4 hours then hyperoxia for 1 hour (Reoxy: reoxygenation). The cells were then incubated in hyperoxia for 10 days. The NAC cultures were the same as the reoxygenated cells except that the medium contained N-acetyl-L-cysteine (NAC) at a concentration of 1 mM. Data represent the mean ± SD of 3 independent experiments. *P < .05 between reoxygenated Fancc^–/– cells and untreated Fancc^–/– or WT NAC or Fancc^–/– NAC samples; **P < .05 between reoxygenated Fancc^–/– cells and any one of all other groups. (B) Expansion of BM cells enriched for HSCs and progenitor cells. The inserted panel shows the initial numbers of the Sca-1⁺ c-kit⁺ and Sca-1^– c-kit⁺ subpopulations in the BM Lin^– cells. WT and Fancc^–/– BM lin^– cells (1 × 10⁵/mL) were treated as described in panel A. At day 5, the cells were counted and then stained with Sca-1–PE and c-kit–APC antibodies and analyzed by flow cytometry. The cell numbers were calculated by multiplication of the total numbers of cells harvested with the percentage of each phenotype of Lin^– cells determined by flow cytometric analysis. The mean ± SD of 3 independent experiments is shown. *Statistical significance between WT and Fancc^–/– samples at P < .01. (C) WT and Fancc^–/– BM lin^– cells described in panel B were counted on day 0 and day 5 after hyperoxic-hypoxic-hyperoxic treatment, stained with Sca-1–PE and c-kit–APC antibodies, and then with annexin V–FITC. Percentages of apoptosis in the 2 subpopulations were analyzed by flow cytometry. Data represent the mean ± SD of 3 independent experiments. (D) Apoptosis in WT and Fancc^–/– BM lin^– cells described in panel D except that analysis was conducted at 0, 4, 8, 16, 24, and 48 hours after hyperoxic-hypoxic-hyperoxic treatment. Data represent the mean ± SD of 3 independent experiments. *P < .05 between reoxygenated Fancc^–/– cells and untreated Fancc^–/– cells. **P < .01 between reoxygenated Fancc^–/– and reoxygenated WT cells. (E) Effect of reoxygenation on BM progenitor activity. The colony-forming cell (CFC) activity of WT and Fancc^–/– BM lin^– cells was evaluated after hyperoxia (Con) only or 2 cycles of hyperoxic-hypoxic-hyperoxic treatments (Reoxy). Data shown represent the number (mean ± SD) of CFU-GMs, BFU-Es, and total number of colonies from 3 independent experiments. *Statistical significance between WT and Fancc^–/– samples at P < .05.