Fig. 1. TNF suppresses HSC/progenitor cell activity. (A) Effect of TNF on HSC frequency. Wild-type (WT) or Fancc^–/– mice were injected intraperitoneally (i.p.) with mouse recombinant TNF in PBS at a dose of 0.1 mg/kg per day for two consecutive days. The mice were then sacrificed 24 hours later and BM mononuclear cells were prepared and stained with antibodies against lineage markers (Lin) and Sca-1-PE and c-Kit-PE-Cy7. Cells were then analyzed by flow cytometry to obtain fractions representing HSCs. The frequencies of LSK cells as a percentage of total BM mononuclear cells are indicated. (B) The effect of TNF on colony-forming activity of WT and Fancc^–/– BM progenitor cells was evaluated using LSK cells isolated from control (PBS-injected) or TNF-injected mice. Data shown represent the mean ± s.d. of colony numbers from three independent experiments; ^*P<0.05. (C) TNF inhibited BM-progenitor proliferation. LSK cells isolated from TNF-injected mice were analyzed for series-plating efficiency of hematopoietic progenitor cells. Data represents the mean ± s.d. of three experiments. (D) TNF inhibited HSC renewal. 2x10⁶ BM cells isolated from control (PBS-injected) or TNF-injected mice (CD45.2⁺) were transplanted together with 1x10⁶ competitor cells from B6.BoyJ mice (CD45.1⁺) into lethally irradiated recipient (B6.BoyJ) mice and long-term engraftment was evaluated 16 weeks after transplantation. Shown are representative flow cytometric data of two independent experiments (n=6). Numbers in the corners indicate percent of events in that quadrant.