Intracellular 4-HNE and GSH levels in EAhy926 endothelial cells. (A,B) Immunofluorescence analysis of the formation of 4-HNE adducts with histidine in EAhy926 cells stained with a 4-HNE specific antibody and counterstained with Hoechst dye to reveal nuclei. Panel A left, untreated cells; panel A right, H2O2-treated cells; panel B left, H2O2-treated cells incubated for 24 hours with fresh medium; panel B right, H2O2-treated cells incubated for 24 hours with medium containing anti-RLIP76 C-ter antibodies. Magnification 1500×, objective 100×, numeric aperture 1.4. (C) Quantitative analysis of 4-HNE adducts by flow cytometry in a representative experiment. Gray histogram, untreated control cells; red histogram, H2O2-treated cells; black histogram, H2O2-treated cells incubated for 24 hours with fresh medium; blue histogram, H2O2-treated cells incubated for 24 hours with medium containing anti-RLIP76 C-ter antibodies. Numbers represent median values of fluorescence intensity. (D) Quantitative time-course evaluation of 4-HNE intracellular content in untreated control cells; in cells incubated for 30 minutes, 6 hours, and 24 hours with anti-RLIP76 C-ter antibodies; in cells treated with H2O2; and in cells treated with H2O2 and then incubated for an additional 30 minutes, 6 hours, and 24 hours in fresh medium or in medium containing anti-RLIP76 C-ter antibodies. Statistical analysis performed by Student t test indicated P < .01 for: control untreated cells versus cells incubated with anti-RLIP76 C-ter antibodies for 30 minutes, 6 hours, and 24 hours; untreated cells versus H2O2-treated cells incubated for 30 minutes and 6 hours in fresh medium; H2O2-treated cells incubated in fresh medium versus H2O2-treated cells incubated for 6 hours and 24 hours with medium containing anti-RLIP76 C-ter antibodies. (E) Quantitative time-course evaluation of GSH intracellular content. Statistical analysis performed by Student t test indicated P < .01 for: untreated cells versus H2O2-treated cells incubated for 30 minutes in fresh medium; untreated cells versus cells incubated for 30 minutes, 6 hours, and 24 hours with anti-RLIP76 C-ter antibodies; H2O2-treated cells incubated in fresh medium versus H2O2-treated cells incubated for 6 hours and 24 hours with medium containing anti-RLIP76 C-ter antibodies. Data reported in panels D and E are the mean plus or minus SD of the results obtained from 3 different experiments.
