Figure 4.

Upregulation of c-FLIP_S is essential for clonogenic survival of NSCLC cells treated with TRAIL. (a and b) NSCLC cells were treated with TRAIL (3 days, 200 ng/ml) and DISC components were analyzed by immunoblot. (c) A549 cells that survived 3 days treatment with TRAIL were untreated (Untr) or treated with CHX (10 μg/ml) simultaneously with freshly added TRAIL (200 ng/ml). Processing of procaspase-8, expression of c-FLIP_S, and formation of cleaved (Cl) PARP-1 were detected by immunoblot. (d) The effect of silencing of c-FLIP_L and c-FLIP_S on TRAIL-mediated activation of procaspase-8 and -3, and cleavage of PARP-1. (e and f) Clonogenic survival of A549 cells transfected with small interfering (si)RNAs targeting c-FLIP_L, c-FLIP_S, and procaspase-8. At 24 h after siRNA transfection, cells were seeded in 6-well plates, and 24 h after the seeding, cells were treated with TRAIL (200 ng/ml). Cell colonies were fixed and counted 14 days after the treatment. Ratio of the number of colonies in TRAIL-treated divided by the number of colonies in TRAIL-untreated samples for each specific siRNA is presented as bars in (f). (g) Processing of procaspase-8 in A549 cells transfected with siRNAs targeting procaspase-8, c-FLIP_L, and c-FLIP_S, and treated with TRAIL (200 ng/ml, 2 h). Cells from the same transfection experiment were used for clonogenic assay. Error bars represent S.E. *P<0.05. FADD, Fas-associated protein with death domain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; scr, scrambled