Moderate Bcl-2 overexpression effectively protects NCI-H460 cells from TRAIL-induced apoptosis.

<p>(A) Expression of FLAG-tagged Bcl-2 in NCI-H460 cells. Equal amounts of whole cell lysates from NCI-H460 wild type and NCI-H460/Bcl-2 were analyzed by immunoblotting. Note that the tagged Bcl-2 protein appears at a somewhat lower molecular weight range as compared to wild-type Bcl-2. Equal loading was confirmed by re-probing the membrane with α-tubulin specific antibody. (B) Detection of Bcl-2 by flow cytometry. Fixed cells were permeabilized and stained with anti-Bcl-2 or corresponding isotype control primary antibody and PE-labeled secondary antibody. Cells were analyzed by flow cytometry. (C) Cytotoxicity assays of NCI-H460 and NCI-H460/Bcl-2 cells using Db-scTRAIL. Cells were treated with increasing concentrations of Db-scTRAIL for 24 h, viable cells were stained with crystal violet and quantified at 550 nm. All values were normalized to values from unstimulated cells. Shown are mean values ± SD calculated from triplicates. Data shown is representative of three independent experiments. (D) Db-scTRAIL induces apoptosis in NCI-H460. Cells were co-treated with Db-scTRAIL and z-VAD-fmk (20 μM) or only the carrier DMSO for 24 h. Viable, apoptotic (Annexin V-FITC-positive) and necrotic (PI-positive) cells were determined after flow cytometric analysis. (E) Db-scTRAIL induces cytochrome c and Smac release into the cytosol in wild-type NCI-H460 cells only. Cells were left untreated or treated with Db-scTRAIL (0.5 nM) for the indicated time periods. Cytosolic extracts were prepared and subjected to western blotting with cytochrome c and Smac specific antibodies. Equal loading was confirmed by reprobing the membrane with α-tubulin antibody. (F) ABT-737 resensitizes NCI-H460/Bcl-2 cells. Cells were preincubated with 2.5 μM ABT-737 alone (filled triangle) or in combination with 50 μM z-VAD-fmk (filled circles) or DMSO as control (open squares) and treated with Db-scTRAIL. Viable cells were quantified by crystal violet staining after 24 h. Data represents mean values ± SD calculated from triplicates and shown is a representative of three independent experiments.