FIG.6.

FLIP is an important mediator of myc-induced sensitization to TRAIL. (A) U2OS cells were transfected with the indicated plasmids. (Left panel) At 48 h later, the cells were collected and immunoblotting was performed. (Center and right panels) Cells were transfected with the indicated plasmids, serum starved 24 h later, and treated with TRAIL for 6 h (50 ng/ml). The cells were collected, stained, and analyzed as shown. (B) HCT116 cells were transfected with the indicated siRNA oligonucleotides and plasmids, collected after 48 h, and analyzed for c-myc and FLIP levels. (C and D) HCT116 cells are resensitized to TRAIL after combined c-myc and FLIP mRNA knockdown. (C) Whole-cell lysates from HCT116 cells transfected and treated with TRAIL for 6 h were collected and analyzed for caspase 8. (D) (Left panel) HCT116 cells were transfected, treated with TRAIL for 6 h, collected, fixed, and stained with propidium iodide. DNA content was measured by flow cytometry, and percentages of cells with sub-G₁ DNA content are shown. (Right panel) TRAIL was added for 4 h, and cells were stained with annexin V and analyzed by flow cytometry. Percentages of cells staining positive for annexin V are shown. (E) More cleaved FLIP and caspase 8 is found at TRAIL DISC in cells with activated myc. WI-38 myc-ER cells were serum deprived for 24 h and treated with 4-hydroxytamoxifen for an additional 24 h or left untreated. The cells were collected, TRAIL was added for 30 min, and DISC immunoprecipitation was performed. (F) U2OS cells are made resistant to c-myc action by introduction of exogenous FLIP. (Upper panel) Immunoblot showing the basal expression of FLIP in U2OS cells stably expressing either a control vector or FLIP under the control of a tetracycline-responsive element. This system is leaky, and a low level of exogenous FLIP expression occurs in the absence of tetracycline. (Lower panel) Sub-G₁ DNA content of serum-deprived cells infected with c-myc-expressing adenovirus for 24 h and treated with TRAIL (200 ng/ml) for 16 h.