FIG. 6.

dRP induces oxidative stress without significantly increasing apoptosis. (A) HUVECs treated with or without 200 μM dRP for 4 h were labeled for 2 h with 20 μM BIAM in anoxic conditions. Thiol oxidation status was determined by protein separation using SDS-PAGE and staining with HRP-streptavidin. Green arrows indicate thiol oxidation, while red arrows represent thiol reduction. β-Actin immunoblotting was used to confirm equal loading. Blots are representative of four independent experiments. (B) VitaBright-43^™ staining was also utilized to measure the level of intracellular reduced thiols. HUVECs were treated with vehicle solution (Tyrode's HEPES buffer) or stimuli with/without NOX inhibitor (200 μM dRP, 10 μM DPI) for 30 min. Cells were costained with VitaBright-43 and propidium iodide and analyzed by image cytometry using the NucleoCounter NC-3000^® system. Plots comparing VitaBright-43 (VB) intensity versus propidium iodide intensity are shown (i). Intracellular thiol oxidation was quantified by counting the percent of cells with VitaBright-43 staining below 10,000 rfu (ii). Statistical analysis was performed by one-way ANOVA with Bonferroni post-test (*p < 0.05, compared with vehicle, n = 6). (B) Cell apoptosis was measured by flow cytometry for PE-Annexin V staining. HUVECs were treated with vehicle (Tyrode's HEPES buffer), 200 μM dRP, or 5 mM diethyl maleate (positive control) for 12 h. FSC/SSC and Annexin V staining histograms from four independent experiments are presented (i). Data analysis is also shown (ii). Data are mean ± SEM, analyzed by one-way ANOVA with Bonferroni post-test (*p < 0.05, compared with vehicle, n = 6). BIAM, biotinyl-iodoacetamide; FSC, forward scattering; HRP, horseradish peroxidase; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SSC, side scattering. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars