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5 cells were mixed with virus in 1.4 ml of media and plated into six-well dishes. HeLa, Saos-2, SW 626, and HT-1080 were infected with 12 plaque-forming units (pfu) per cell; IMR-90, WI-38, HS68, and MRC-5 with 36 pfu/cell; MEFs with 100 pfu/cell; and B and T cells with 360 pfu/cell. We have found that long-term (>4 days) adenovirus infection inhibits cell growth and alters the expression of cell-cycle markers. For this reason, data were collected within 96 hours after infection. The adenoviral growth inhibition prevented the analysis of a possible senescence response to TRF proteins.
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ΔM as compared to noninfected control cells.
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5 cells were fixed in 70% ethanol, stained with propidium iodide, and analyzed using a Becton-Dickinson FacsScan and CellQuest software.
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2 accumulation observed in HeLa cells infected with AdTRF1 and AdTRF2 appears to be a HeLa-specific phenotype because it does not occur in HT-1080 (2), WI-38, and IMR-90
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2 accumulation observed in HeLa cells infected with AdTRF1 and AdTRF2 appears to be a HeLa-specific phenotype because it does not occur in HT-1080 (2), WI-38, and IMR-90.
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Protein (30 μg) extracted in 50 mM Hepes-KaOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, and 0.5% NP-40 was fractionated on SDS-polyacrylamide gels, transferred to nitrocellulose membranes, blocked in phosphate-buffered saline (PBS) containing 10% milk and 0.1% Tween 20, and incubated in PBS with 0.1% milk and 0.1% Tween 20 with the following antibodies: anti-p53 (Santa Cruz DO1), anti-Cydin D1 (Santa Cruz R124), anti-Bax (Santa Cruz B-9), anti-p21 (Santa Cruz F-5). anti-FLAG (Kodak M2), or anti-myc 9E10. Blots were washed (three times) in PBS with 0.1% milk and 0.1% Tween 20 and incubated with horseradish peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse (Amersham). Bound antibodies were detected using the ECL kit (Amersham).
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We thank T. Jacks and R. DePinho for MEFs and helpful discussion: N. Dyson, C. Sherr, C. Prives, and K. Kuchler for antibodies; D. Unutmaz and M. Alberts for primary human T cells; T. Jenuwein for CMV-myc TRF1; F. Isdell for expert assistance in the FACS analysis; and C. Price and members of the de Lange lab for their insights. Supported by the Human Fron-tiers Science Program (J.K.), Merck (D.B.), and grants from the New York Community Trust, Rita Allen Foundation, and NIH (GM49046) (T.d.L).
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