Fig 6.

Death ligand elimination assay. (A and B) WISH cells were transfected with cFLIP siRNA or mock and treated with YNS at 1 nM, TNF-α at 25 ng/ml, 1% Fas ligand, TRAIL at 25 ng/ml, Z-VAD at 100 μM, and anti-TNF-α/Fas TRAIL fused protein at 1 μg/ml for 72 h prior to determination of cell density (A) and for 24 h for the caspase-3/7 activity assay (B). Data are represented as means ± standard errors of the means. (C and D) WISH cells were transfected with siRNA targeting cFLIP, RIPK1, cIAP1, or cIAP2 or not transfected (control) following treatment with 1 nM YNS with or without Z-VAD, 1 μg/ml anti-death ligand mix, or 63 μM necrostatin-1 (Nec-1) for 48 and 72 h. Cells were stained with annexin V and PI to determine apoptosis. Data are represented as means. Multiple repeats and experiments have shown an average error of ±10% for the results. (E and F) WISH cells were transfected with siRNA targeting the indicated genes following treatment with 1 nM YNS for an additional 72 h to determine cell density (E) or 24 h for caspase-3/7 activity (F). Caspase activity is relative to untreated cells.