Recombinant human trail apo2 ligand

The antibodies and chemicals were obtained from the following resources; anti-PARP (#556362), anti-XIAP (#610716), anti-FADD (#610399), anti-RIP1 (#610459), anti-p53 (#554147) and anti-p21 (#556430) antibodies (BD Biosciences, San Diego, CA, USA); anti-caspase-3 (#9662), anti-caspase-8 (#9746), anti-caspase-9 (#9508) and anti-Bid (#2002) antibodies (Cell signalling Technology, Beverly, MA, USA); anti-caspase-10 (M059-3) antibody (MBL, WOBURN, MA, USA); anti-Bcl-X_S/L (sc-271121), anti-survivin (sc-17779), anti-TRAF2 (sc-876), anti-GFP (sc-9996) and anti-HA (sc-805) antibodies (Santa Cruz, CA, USA); anti-c-FLIP (ALX-804-961) antibody (Enzo Life Sciences, Farmingdale, NY, USA); anti-cIAP1/2 (#07-759) antibody (Upstate Biotech, Waltham, MA, USA); anti-DR5 (#ab181846) antibody (Abcam, Cambridge, UK); anti-DR4 (NB100-56528) antibody (Novus, Centennial, CO, USA); anti-TurboGFP (PA5-22688) antibody (Thermo scientific, Waltham, Massachusetts, USA); anti-actin (A2066), anti-flag (F3165, 1:2,000 dilution) antibodies (Sigma-Aldrich, St. Louis, MO, USA). the pan caspase inhibitor Z-VAD-FMK, MG-132, TPCA-1 (Calbiochem, San Diego, CA, USA); recombinant TNF (R & D Systems, Minneapolis, MN, USA); recombinant human TRAIL/Apo2 ligand (Peprotech, Rocky Hill, NJ, USA).

Human MDA-MB-231 and MCF-7 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Leibovitz’s L-15 medium, RPMI-1640 medium, Fetal Bovine Serum (FBS) and Penicillin-streptomycin Cocktails were obtained from Thermo Scientific (Rockford, IL). Suberanilohydroxamic acid (SAHA) was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant human TRAIL/Apo2 Ligand was from Peprotech (Rocky Hill, NJ). Muse Cell Cycle kit, Muse Annexin & Dead Cell kit, and Muse Count & Viability kit were from Millipore (Darmstadt, Germany). Human Apoptosis Antibody Array kit was purchased from R&D Systems (Minneapolis, MN). High Pure RNA Isolation kit, Annexin-V-FLUOS staining kit and Transcriptor First Strand cDNA Synthesis kit were obtained from Roche Diagnostics GmbH (Mannheim, Germany). Exprofile Human Cell Cycle Tox and Cancer Related Gene qPCR Array kit and Exprofile Human EGF/PDGF Signaling Related Gene qPCR Array kit were obtained from Genecopoeia (Rockville, MD). Power SYBR Green PCR Master mix, Calcein-AM dye, RIPA Cell Lysis buffer and BCA Protein Assay kit were from Life Technologies (Austin, TX). CellTiter 96AQueous One Solution Cell Proliferation Assay kit was obtained from Promega (Madison, WI). Protease inhibitor and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

The Human NSCLC cell lines NCI-H1975, HCC827 and H358 were obtained from the American Type Culture Collection (ATCC), while PC9 and A549 were obtained from Sigma. These cell lines were chosen based on their expression of mutated genes which are clinically relevant and represent the common heterogeneity of the disease. Cells identity was confirmed by STR analysis. All cells were cultured at 37° C in a humidified 5% CO₂ incubator. H1975, PC9, HCC827 and H358 cells were grown in RPMI-1640 medium (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Perbio Science), 2 mM L-glutamine and 25 mM HEPES. A549 cells were grown in DMEM supplemented with 10% heat inactivated FBS, 2 mM L-glutamine and 25 mM HEPES. Stock Solution (10 mM) of RO3280 (Selleckchem) were prepared in dimethyl sulfoxide (DMSO) and stored at −80° C, further diluted in fresh medium prior to each experiment with the final concentration of DMSO less than 0.1%. Recombinant human TRAIL/Apo2 ligand was purchased from Peprotech and diluted in phosphate buffer saline (PBS).