Figure 5
TRAIL-induced cell death in R1 cells involves the generation of reactive nitrogen species. (a) WT and R1 cells were treated with 50 ng/ml of TRAIL for 2 and 4 h. Cells were subsequently harvested and analyzed by flow cytometry for ROS production using redox-sensitive probe DCFH-DA. (b) R1 cells were preincubated with 50 μM FeTPPs for 1 h followed by 50 ng/ml of TRAIL for 2 or 4 h. Cells were subsequently harvested and analyzed by flow cytometry after loading with DCFH-DA. (c) Cells were treated as above and loaded with NO-specific probe DAF or (d) MitoSox before flow cytomteric analysis. Antimycin A (AA) was used as a positive control for mitochondrial O2−. Data are shown as mean±S.D. of fold differences of fluorescence from untreated cells for at least three independent experiments. (e) Western blot analysis of iNOS following treatment of WT and R1 cells with 50 and 100 ng/ml of TRAIL. (f) R1 cells were preincubated with FeTPPs (50 μM) for 1 h followed by 24 h of treatment with 50 ng/ml of TRAIL. Cell viability was determined by MTT assay and expressed as % of untreated control cells. *P<0.05 compared with 50 ng/ml of TRAIL alone and #P<0.05 compared with 100 ng/ml of TRAIL alone. Data shown are mean±S.D. of at least three independent experiments. (g) Western blot analysis of iNOS in R1 cells, pretreated with 50 μM ZVAD for 1 h followed by 50 ng/ml of TRAIL for 8 h. (h) WT and R1 cells were pretreated with 50 μM ZVAD for 1 h followed by 50 ng/ml of TRAIL for 2 and 4 h. Cells were harvested and analyzed by flow cytometry for intracellular ROS production with DCFH-DA. Data shown are representative of at least three independent experiments. (i) R1 cells were preincubated with FeTPPs for 1 h followed by 24 h of treatment with 50 ng/ml of TRAIL and whole-cell lysates were subjected to western blot analysis for the assessment of caspase 3 and 8 processing as well as PARP cleavage. GAPDH was used as loading control
