Fig. 3.
GW-induced decrease in intracellular O2•-downregulates cFLIP. (A) cFLIP expression was verified in lysates from CEM/Neo and CEM/Bcl-2 cells cultured in normal growth medium or GW medium using anti-cFLIP by Western blot analysis; anti-β-actin was used as loading control. (B) M14TF4 cells were pretreated with PMA (62.5 ng/ml) or paraquat (50 μM) for 1hr followed by 5 ng/ml of TNFα exposure in culture medium or GW medium for 18 hrs and cell survival was assessed by MTT assay as described in Material and Methods. (C) Western blot analysis for cFLIP expression in M14TF4 cells cultured in normal or GW medium; anti-β-actin was used as loading control. (D) CEM/BCL2 cells were transfected with siNeg or siFLIP using RNAiMAX and 48 hrs post transfection incubated with anti-Fas (0.25 μg/ml for 18 hrs) with or without pre-treatment with PMA (62.5 ng/ml for 1hr) in normal growth medium or GW medium or high glucose medium (HG; 20 mM glucose). Caspase-8 activity was determined using a fluorometric assay as described in Materials and Methods. (E) Cell viability was assayed by the MTT assay and presented as % survival relative to untreated cells. Where applicable, data shown are Mean ± S.D. of three independent experiments and P values (** <0.01; *** <0.005) were calculated by Ordinary one-way ANOVA using GraphPad Prism.