Fig. 6.

Increase in O₂^•-amplifies cFLIP promoter activity distinctly in contrast to H₂O₂. (A) M14PIRES and M14RacV12 cells were transfected with a luciferase-tagged plasmid containing the cFLIP promoter for 24 hrs and then treated with PMA (62.5 ng/ml) or DPI (5 μM) for 1hr and promoter activity was measured by luciferase reporter assay. Results are expressed as fold change in luciferase activity. (B) HeLa cells carrying the cFLIP promoter construct were cultured in normal growth medium, GW medium or in the presence of 2DG and cFLIP promoter activity was assessed by the luciferase reporter assay. (C) HeLa/Neo cells were treated with DDC (100 μM and 200 μM) for 2 hrs while HeLa/Bcl-2 cells were treated with H₂O₂ (100 μM–400 μM) for 2 hrs and whole cell lysates were subjected to Western blot analysis using anti-cFLIP and anti-β-actin. (D) CEM/Neo cells were pretreated with H₂O₂ (50 μM–400 μM) for 1hr followed by anti-Fas (0.25 μg/ml) for 18 hrs and cell survival was determined by the MTT assay. (E) cFLIP expression in whole cell lysates from the cells in D was determined by Western blot analysis using anti-cFLIP as described in Materials and Methods; anti-β-actin was used as loading control. Data in A, B and D are shown as Mean ± S.D. of three independent experiments and P values (* <0.05; *** <0.005) were calculated by Ordinary one-way ANOVA using GraphPad Prism.