Amplifications were performed in triplicate according to the cycling protocol provided by the manufacturer. Gene expression was expressed as 2-ΔΔ(Ct) [18], where Ct is cycle threshold,

Δ(Ct) = Ct of tested gene – Ct of GAPDH; ΔΔ(Ct) = Δ(Ct) of sample 1-Δ(Ct) of sample 2. Western blot analysis The mouse anti-human Fas (cat. sc-74540), GST-π (cat. sc-58368) and rabbit anti-human ERCC1(cat. sc-10785) antibodies and horseradish peroxidase(HRP)-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G (IgG) were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). 5 × 106 H446/CDDP Cells were seeded into 100 mm plates, Ro 61-8048 incubated for 24 h at 37°C, and then transfected with 50 MOI of adenoviruses. On post-transfection day 3, H446/CDDP, H446/CDDP/Fas, and H446/CDDP/empty cells were washed three times with cold phosphate buffered saline (PBS) and then lysed in RIPC buffer (0.5

M NaCl, 0.5% NP-40, 20 mM Tris-HCl pH 8, 1 mM PMSF). The protein levels were determined using an ECL kit ((Amersham Pharmacia, Uppsala, Sweden). Total cellular proteins were diluted 2-fold into SDS-PAGE loading buffer (NEB). The samples were heated to 95°C for 5 min before an aliquot of 20 μl of each diluted assay sample, containing approximately 50 ug of total protein, was loaded onto a 6-12% Tris-glycine polyacrylamide gel (Invitrogen). Proteins Selleckchem PSI-7977 were resolved by SDS-PAGE and then transferred to a 0.45 μm nitrocellulose membrane (Whatman). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) supplemented with 0.2% Tween Rolziracetam 20 and 0.05% Triton X-100 (TBSTT). The membrane was probed with the primary antibody at 1:700 dilution in TBSTT supplemented

with 2% nonfat dry milk. After an overnight incubation at 4°C, the membrane was washed and incubated at room temperature for 2 h with a goat anti-rabbit or mouse HRP-linked IgG antibody (1:700 dilution in TBSTT with 2% dry milk). Binding of the antibody was www.selleckchem.com/products/gdc-0068.html detected by chemiluminescence with the Phototope-HRP Western Blot Detection System (CST). In vitro drug sensitivity assay Drug sensitivity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, on post-transfection day 3, the transfected cells and control cells were seeded into 96-well plates with 103 cells per well and incubated overnight. Cells were then incubated with CDDP in different concentrations (5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 μg/ml). After 72 h of incubation, 20 μl of 5 mg/ml MTT (Sigma Chemical Co., St Louis, MO) in PBS was added to each well, followed by incubation for 4 h at 37°C. The formazan crystals were dissolved in 50 μl of dimethyl sulfoxide (DMSO). The optimal density was determined with microculture plate reader (Becton Dickinson Labware, Lincoln Park, NJ) at 570 nm.