Effects of anti-RLIP76 C-ter antibodies on human microvascular primary cells. (A) Flow cytometric analysis of HMVEC-L cells, either untreated or H₂O₂-treated cells, after surface staining with antibodies to RLIP76. Mean plus or minus SD of the results obtained from 3 different experiments. *P < .01 by Student t test. (B,C) Quantitative flow cytometric analysis of 4-HNE adducts and GSH intracellular content in: untreated control cells, cells treated with H₂O₂ for 30 minutes, cells incubated with anti-RLIP76 C-ter antibodies for 24 hours, and cells treated with H₂O₂ for 30 minutes and then incubated for an additional 24 hours in medium containing anti-RLIP76 C-ter antibodies. Data reported in panels B and C are the mean plus or minus SD of the results obtained from 3 different experiments. Student t test indicated P < .01 for: control untreated cells versus H₂O₂-treated cells; untreated cells versus cells incubated with anti-RLIP76 C-ter antibodies for 24 hours; and untreated cells versus H₂O₂-treated cells incubated for 24 hours with medium containing anti-RLIP76 C-ter antibodies. (D) Dot plots from a representative experiment performed 48 hours after different treatments. Numbers represent the percentage of annexin V single positive (early apoptosis, bottom right quadrant) or annexin V/PI double positive cells (late apoptosis, bottom right quadrant). (E) Flow cytometric analysis of apoptosis after double staining with annexin V/propidium iodide of untreated control cells; cells treated with H₂O₂ and then incubated at different time points with fresh medium or with medium containing anti-RLIP76 C-ter antibodies. Cells were also treated for the same times with anti-RLIP76 C-ter antibodies given alone. Results obtained from 3 independent experiments are reported as mean plus or minus SD. Student t test indicated P < .01 for: control untreated cells versus cells incubated for 24 and 48 hours with anti-RLIP76 C-ter antibodies, and cells treated with H₂O₂ and then incubated in fresh medium versus cells treated with H₂O₂ and then incubated for 24 and 48 hours with medium containing anti-RLIP76 C-ter antibodies. (F) Quantitative flow cytometric analysis of apoptosis in cells treated with anti-RLIP76 C-ter antibodies for 24 and 48 hours pretreated or not with zVAD or α-TCPH as indicated in “Methods.” As control, cells were also treated at the same time points with zVAD or α-TCPH given alone. Results obtained from 3 independent experiments are reported as mean plus or minus SD. (G) Quantitative flow cytometric analysis of the JNK activation state obtained with a polyclonal antibody specific for active form of JNK (pT183/pY185) in cells treated with anti-RLIP76 C-ter antibodies in the presence or absence of α-TCPH. * represents P < .01 by Student t test.