Raulet_Figure S4

1. A Ion. Rad. (40 Gry 137Cs) Ara-C (10 µM) Cisplatin (10 µg/ml) UV-C (30 J/m2) 50 mM NaCl 5-FU (10 µM) Chloroquine (32 µg/ml) HU (5mM) Mitomycin C (30 µM) NKG2D ligand expression B ATM RNAi GFP+ GFP- Aphidicolin (4 M) UV-C (30 J/m2) Ion. Rad. (40 Gry 137Cs) 50 mM NaCl NKG2D ligand expression Supplementary Figure S4. ATR/ATM inhibitors prevent NKG2D ligand upregulation. A) Caffeine, an inhibitor of ATR and ATM inhibits ligand upregulation. Subconfluent cultures were treated with caffeine beginning 1 hour before indicated treatments. Cell surface ligand expression was determined 16 hours later. Treated cells incubated with 10 mM caffeine (thin line) were compared to treated cells incubated with no inhibitor (thick solid line), or to treated cells stained with control tetramers (filled histograms). B) ATM siRNA inhibits ligand upregulation induced by ionizing radiation and hypotonic conditions. Fibroblasts were transduced with retroviral vectors encoding GFP and ATM siRNA (dashed lines in histograms) or GFP and control siRNA (solid lines), cultured for 4 days, splitting as necessary, and replated for 1 day before treatment with aphidicolin, 40 Gry, UV-C or 50 mM NaCl. After 16 hours, cells were harvested and examined for NKG2D ligand expression, gating on transduced (GFP+) and untransduced (GFP-) cells from the same cultures. Dotted line: untreated cells stained with NKG2D tetramers. Filled histogram: aphidicolin-treated cells stained with control tetramers. As determined by real-time RT-PCR, ATM transcripts in ATM siRNA transduced cells were reduced to 47% + 3% of the level in untransduced (GFP-) cells in the same culture, or 44% + 3% of the level in control siRNA transduced cells (mean + SD of three determinations). Because of impurities in the sorted populations, the knockdowns may be more efficient than indicated. Raulet et al (MS 2004-12-27903B-Z) Raulet_Figure S4