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Fig. 3. TNF induces premature senescence in HSC/progenitor cells. (A) WT or Fancc^–/– mice were injected i.p. with two doses of TNF (100 µg/kg per day) for 2 consecutive days. The mice were then sacrificed 24 hours later and BM LSK cells were isolated and stained for SA--gal. The bar graph shows the percentages of the cells stained positive for SA--gal; cells were quantified by counting >100 cells in random fields on a slide for each of three independent experiments. The data represent the mean ± s.d. of three independent experiments; ^*P<.05. (B) Senescence HP1- staining. BM LSK cells isolated from PBS- or TNF-treated WT and Fancc^–/– mice were stained for HP1-. DNA was then labeled with DAPI. (C) Frozen (SA--gal; magnification, 20x) or paraffin-embedded (HP1-; magnification, 40x) BM sections of PBS- or TNF-treated WT and Fancc^–/– mice were subjected to SA--gal (left) or HP1- (right) staining. (D) Paraffin-embedded spleen sections of PBS- or TNF-treated WT and Fancc^–/– mice, stained with antibody against the proliferation marker Ki-67 (magnification, 20x). (E) BM LSK cells of TNF-treated Fancc^–/– mice showed strong immunostaining for p53 and p16^Ink4a. The BM LSK cells isolated from PBS- or TNF-treated WT and Fancc^–/– mice were stained with the antibodies against p53 and p16^Ink4a and then counterstained with DAPI. The bar graph shows the percentages of the cells stained positive for p53 and p16^Ink4a; cells were quantified by counting >100 cells in random fields on a slide for each of three independent experiments; ^*P<0.05.

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