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Fig. 6. TNF-induced senescence of HSCs and progenitor cells is associated with oxidative DNA damage and genomic instability. (A) WT or Fancc^–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. NAC (1 mg/mouse per day) was administered 30 minutes before and after each TNF injection. The mice were then sacrificed 24 hours later and BM cells from individual mice were analyzed for DNA strand breaks in a the comet assay. Larger tails represents higher levels of DNA damage. For each treatment, at least 100 cells were scored from random sampling. Data are expressed as the mean ± s.d. of two independent experiments, each with three mice (six mice per group). (B) WT or Fancc^–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. NAC (1 mg/mouse/day) was administered 30 minutes before and after each TNF injection. The mice were then sacrificed 24 hours later and BM LSK cells were isolated and stained for the oxidative DNA damage marker 8-oxo-deoxyguanosin (8-oxodG). The bar graph shows the percentages of the cells stained positive for 8-oxodG and quantified by counting >100 cells in random fields on a slide for each of two experiments with total 6 mice. (C) Examples of metaphase chromosomes prepared from TNF-treated WT and Fancc^–/– BM cells. Arrows indicate aberrant chromosomes. (D) WT or Fancc^–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. NAC (1 mg/mouse/day) was administered 30 minutes before and after each TNF injection. Mice were sacrificed 24 hours later and BM LSK cells were then isolated and stained for p53^Ser20 and H2AX. Bar graphs show the percentages of the cells stained positive for p53^Ser20 and H2AX. Cells were quantified by counting >100 cells in random fields on a slide for each of two experiments with total six mice. ^*P<0.05. (E) Kinetics of DNA repair of oxidative DNA damage and DNA strand breaks. BM cells from WT and Fancc^–/– mice were treated with or without H₂O₂ (100 µM) for the indicated time periods, and protein extracts were prepared and analyzed by immunoblotting with antibody against phosphorylated p53^Ser20 (p53^Ser20) and anti-H2AX and anti-actin antibodies. Extracts were also prepared from cells 2 hours (2-) after H₂O₂ withdrawal after the cells had been treated with H₂O₂ for 4 hours.

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