<p>(A) Downregulation of XIAP by siRNA treatment. Cells were left untreated (UT) or were transiently transfected with non-targeting (siControl) or siRNA specific for XIAP (siXIAP). Protein expression was determined 48 h post transfection from whole cell lysates <i>via</i> immunoblot assay using an XIAP specific antibody. As control c-IAP1 and survivin were included. (B) and (C) NCI-H460 wild type (B) and NCI-H460/Bcl-2 cells (C) had been pretreated as in (A) were treated with serial dilutions of Db-scTRAIL (open symbols). NCI-H460/Bcl-2 cells transfected with siXIAP were incubated with 50 μM z-VAD-fmk and 1 nM Db-scTRAIL (filled triangle). After 24 h viable cells were stained with crystal violet. All values were normalized to those from unstimulated cells. The experiment was performed in triplicates and the data shown are representative of three independent experiments. (D) NCI-H460/Bcl-2 cells had been pretreated as in (A) were treated with Db-scTRAIL (1 nM) for 2 and 4 h and analyzed by immunoblotting using antibodies for cleaved caspase-8, -9 and -3. Tubulin-α was used as loading control. Blot shown is representative of three independent experiments.</p
