Effect of vitamin C on caspase-8 activity. (A) U937 cells were loaded with 13 mM vitamin C prior to FASL treatment. The cell lysates were analyzed for caspase-8 activity. Error bars represent the SD of triplicate values. (B) A Western blot was performed to analyze the expression of pro—caspase-8 in U937 cells loaded with 13 mM vitamin C (arrow, upper panel). Detection of β-tubulin was used as control for protein loading (arrow, lower panel). (C) Monocytes were loaded with 14 mM vitamin C prior to FASL treatment and the cell lysates were analyzed for caspase-8 activity. The results are the mean of triplicate samples expressed as the percentage increase in caspase activity compared with untreated cells and represent 3 (U937) or 2 (monocyte) experiments. (D) The activity of recombinant caspase-8 (1.25 U/well) with and without vitamin C was determined as detailed in “Materials and methods.” These results are expressed as the percentage of activity in relation to control wells (without vitamin C) and are representative of 3 independent experiments. Asterisks indicate statistically significant differences (P = .0005) in caspase activity of cells loaded with vitamin C before challenge with FASL.