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Fig. 3. Effect of metabolic inhibitors on ribonuclease-mediated cytotoxicity. (A) Cells were incubated with NaN3 (10 mM) and 2-deoxyglucose (50 mM) or PBS control for 1 hour at 37°C. ONC (10 µM) or K7A/G88R RNase A (25 µM) was then added, and cells were incubated for an additional 6 hours. DNA synthesis was measured as described in Materials and Methods. (B) Cells were incubated with NaN3 (10 mM) and 2-deoxyglucose (50 mM). BODIPY~RNase A (1 µM) was then added, and cells were incubated for an additional 30 minutes at 4°C. Cells were pulsed at 37°C for 30 minutes, and the fluorescence of BODIPY~RNase A was visualized directly.