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[PMC free article] [PubMed] [Google Scholar] 22. of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, = RAD51 Inhibitor B02 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors. INTRODUCTION The identification and validation of novel drug targets continues to be a major bottleneck in drug development. The advent of high-throughput analysis of gene function using short interfering RNA (siRNA)-based RAD51 Inhibitor B02 screens provides an efficient means to predict novel functions of gene products in cellular signaling pathways and to identify potential drug targets (1, 2). A portion of the human genome has been identified that encodes proteins with functions that are predicted to be or are targets for drugs used for human diseases; this socalled druggable genome comprises between 3,000-10,000 genes (3, 4). The products of these genes include protein classes such as kinases, G-protein coupled receptors (GPCRs), phosphatases, proteases, and ion channels (3). It has been hypothesized that by focusing on the druggable genome, the likelihood of finding useful drug targets for human diseases could be increased. The identification and development of radioprotectors (administrated prior to irradiation) and mitigators (administrated after irradiation but before clinical syndromes are detected) is of considerable importance because of the possible applications in clinical radiotherapy, after accidental exposure to radiation, and in radiation counterterrorism (5). In the present study, we tested the hypothesis that well-characterized drugs might have radioprotective effects. Using a synthetic protection assay, we screened siRNAs that were nontoxic for protective effects against a cytotoxic dose of ionizing radiation. We used a siRNA library comprising 16,560 unique siRNA sequences targeting 5,520 genes (Supplementary Table 1) that are known to encode gene transcripts considered to be actual or potential drug targets or to modify disease. We discovered that the commonly used hypoglycemic agent glyburide protected mice against total-body irradiation. These results illustrate the power of a combined approach of high-throughput siRNA screening and conventional cell-based assay to identify new radioprotectors. MATERIALS AND METHODS Reagents The DharmaFECT 2 transfection reagent and 5 siRNA resuspension buffer were from Dharmacon (Lafayette, CO). The RAD51 Inhibitor B02 cell Titer-Blue Cell Viability Assay was from Promega (Madison, WI). The 384-well tissue-culture treated microtiter plates were from Greiner Bio-One (GmbH, Frickenhausen, Germany). OptiMEM, MEM and FBS were from Invitrogen (Carlsbad, CA). The RAD51 Inhibitor B02 Silencer Druggable Genome siRNA Library (Version 1.1) was from Ambion (Austin, TX). The Annexin V kit was from Biovision (Mountain View, CA). The lactate dehydrogenase (LDH) viability kit was from Sigma (St. Louis, MO). Rabbit polyclonal anti-ABCC8 antibody was from Abcam (Cambridge, MA). Mouse monoclonal anti-actin antibody was purchased from Sigma (A3853). Cell Culture Human glioblastoma T98G and U-87 MG cells (American Type Culture Collection, Manassas, VA) were maintained in MEM supplemented with 2 mglutamine, 10% FBS and penicillin-streptomycin. Human primary astrocytes were purchased from ScienCell (Carlsbad, CA) and maintained according to the manufacturers instructions. Normal human lung epithelial BEAS-2B cells were from American Type Culture Collection and were cultured in a serum-free bronchial epithelial growth medium (Lonza, Walkersville, MD). The 32D cl3 mouse hematopoietic progenitor Rabbit Polyclonal to SEMA4A cell line, dependent for growth upon interleukin 3 (IL-3), has been described previously (6). 32D cl3 cells were passaged in fresh RMPI 1640 medium containing 10% FBS, 1% glutamine, penicillin-streptomycin and 15% WEHI-3 conditioned medium as a source of IL-3. High-Throughput siRNA Delivery by Reverse Transfection Human glioblastoma T98G cells were reverse transfected (7) with the siRNA library in 384-well plate at a final concentration of 20 nfinal concentration) or scrambled control siRNA (Ambion Silencer Negative Control no. 3 siRNA, 20 nfinal concentration) using DharmaFECT 2 transfection reagent according to the manufacturers instructions. After 48 h incubation, harvested cells were lysed on ice for 30 min in RIPA buffer (50 mTris-HCl, pH 7.5, 150 mNaCl, 1% IGEPA, 0.5% sodium deoxycholate, 0.1% SDS in the presence of proteinase inhibitors) and then centrifuged at 10,000for 5 min. The resulting supernatants were subjected to 8% SDS-PAGE and then transferred to a nitrocellulose membrane. The membrane was cut into two sections according to the position of molecular markers and then probed with anti-ABCC8 and anti-actin (loading control), respectively, followed by horseradish peroxidase-coupled detection. Validation of siRNA Detected Targets with a Cell-Based Assay For target validation experiments, cells RAD51 Inhibitor B02 were seeded in 35-mm dishes at a density of 1 1 105/dish and allowed to attach overnight. Cells were incubated with drugs in fresh culture medium at the.

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