2 siRNA

1. DNAPK siRNA A 2 siRNA Control C ii) 120 i) B AA8 control 100 CHOV3 DNAPK-/- 80 [35S]-Met Incorporation % [35S]-Met Incorporation % 60 40 20 0 Time (h) Time (h) A reduction in DNA-PKcs by RNAi also prevents translational inhibition A) Western analysis of DNA-PKcs levels in HeLa cells following transfection with control siRNA (C2) or a siRNA complementary to DNA-PKcs mRNA. There is a reduction to approximately 50% of the expressed protein in the presence of the siRNA. B) Protein synthesis rates were determined by liquid scintillation counting of newly incorporated [35S] Methionine in mock treated cells and UVB irradiated cells (275 J/m2). The data show that in the cells that have reduced expression of DNA-PKcs by RNAi (i) or in knockout mice (ii) there is a reduction in the degree of translational shut down following UV exposure. Control GM00200 120 Seckle ATR-/-l GM18366 100 80 [35S]-Met Incorporation % 60 40 20 0 0 4 Time (h) C) Protein synthesis rates were determined by liquid scintillation counting of newly incorporated [35S] Methionine in mock treated cells and UVB irradiated cells (275 J/m2). The data show that in the seckle cells that have reduced expression of ATR (Stokes et al 2007) there is a small reduction (15%) in the degree of translational shut down following UV exposure. Supplementary 5