Lc 2 ad

The LCN1, derived from large cell neuroendocrine carcinoma, was established in our laboratory [15 (link)]. The LC-2/ad and A549, both derived from a lung adenocarcinoma, were purchased from the RIKEN BioResource Center (Ibaraki, Japan) and Japanese Cancer Research Resources Bank (Tokyo, Japan), respectively. These cells were grown in RPMI-1640 medium (SIGMA, Steinheim, Germany) supplemented with 10% fetal bovine serum (FBS; Biowest, Miami, FL, USA), 100 units/ml of penicillin, and 100 μg/ml of streptomycin (GIBCO, Auckland, New Zealand). After harvesting and washing twice with phosphate-buffered saline without divalent ions (PBS-), sub-confluent cells were stored at -80°C for proteomic analysis and fixed in 10% formalin and embedded in paraffin for immunohistochemistry. LCN1 cells were also AMeX-fixed [16 (link)] for immunohistochemical screening. The SP2/O-Ag14 cells derived from a mouse myeloma were purchased from the RIKEN BioResource Center, and were grown in RPMI-1640 medium supplemented with 1×8-azaguanine (50× Hybri-Max, SIGMA), 10% FBS, 100 units/ml of penicillin, and 100 mg/ml of streptomycin (GIBCO).

The human NSCLC cell lines RERF-LC-AI, RERF-LC-KJ, and LC2/Ad were obtained from Riken BRC through the National Bio-Resource Project of the MEXT (Tsukuba, Japan). The A549 cells were purchased from both Riken BRC and American Type Culture Collection (Manassas, VA), while the PC–9 cell line was obtained from IBL cell bank (Gunma, Japan). The genotypes of all cell lines were identified with STR Identifier (Applied Biosystems) or PowerPlex 16 STR system (Promega). All the cell lines were maintained in culture in RPMI 1640 medium with 2mM L-glutamine (Invitrogen) supplemented with 10% FBS (Sigma-Aldrich) (A549, RERF-LC-AI, RERF-LC-KJ and PC–9) or 15% FBS (LC2/Ad) and 50 U/ml penicillin streptomycin (Sigma-Aldrich) at 37°C in a humidified atmosphere with 5% CO₂. For cell culture work, Gefitinib (Cayman, #13166), Docetaxel (Enzo Life Sciences, BML-T129), LY294002 (Cayman, #70920), PD98059 (Cayman, #10006726) and KU55933 (Tocris Bioscience, #3544) were dissolved in DMSO (Sigma-Aldrich). Gemcitabine (Tocris Bioscience, #3259), Pemetrexed (Santa Cruz, #sc–219564), Vinorelbine (Santa Cruz, #sc–216059), Caffeine (Tocris Bioscience, #2793) and N-acetyl-L-cysteine (NAC) (Sigma-Aldrich, #A9165) were dissolved in PBS (-).