Vitamin C prevents mitochondrial damage induced by FAS-R ligation.(A) U937 cells were loaded with 13 mM vitamin C prior to 3 hours of treatment with FASL (200 ng/mL) and were stained with 40 nM DiOC(6)(3) for 15 minutes at 37°C. Control cells were treated with 0.2 μM Z-IEDT-FMK. DiOC(6)(3) fluorescence was analyzed by flow cytometry. The intensity fluorescence of DiOC(6)(3) is shown on the x-axis, and the forward scatter is represented on the y-axis. The numbers in the figure indicate the frequency of the population with low intensity of DiOC(6)(3) fluorescence for each treatment and are representative of 7 independent experiments (except for the experiment with Z-IEDT-FMK, which was performed twice). (B) Cells cultured under the conditions described in panel A were stained with annexin-PI to assess frequency of apoptosis. These results are the mean of 3 independent experiments, performed in duplicates, and are presented as the fold increase in apoptosis over untreated control cells. (C) U937 cells were loaded with approximately 13 mM vitamin C prior to treatment with 200 ng/mL FASL. Cytosolic cell extracts were obtained and analyzed by ELISA for the presence of Cyt C. The results show the normalized Cyt C content for each experimental condition in arbitrary units based on the Cyt C cytosolic content of control cells and represent 2 independent experiments. Asterisk indicates statistically significant differences (P = .039) using the Student t test between control and cells loaded with vitamin C before challenge with FASL. Error bars represent the SD of triplicate values.
