hown within the figures.Survival assays utilizing siRNA transfected HeLa cells were performed 48 h immediately after the transfection. Cells (transfected with either the handle or two various RECQ1 siRNAs) had been seeded in quadruplicate at a density of 300 cells/well in 96-well plates, allowed to adhere for 146 h, and subsequently exposed to rising doses of IR working with Gammacell 40 (Nordion International, Inc.), a 137Cs supply emitting at a fixed dose price of 0.82 Gy/ min. To identify CPT sensitivity, cells had been exposed constantly to rising concentrations of CPT (Sigma, St. Louis, MO). Following treatment, cells have been permitted to develop at 37uC for five days in 5% CO2. Plates have been frozen at 280uC. Total DNA was quantified employing CyQuant (Molecular Probes, Eugene, OR) and compared with untreated controls as an indication of cell growth as described [15,16]. Quantification of DNA working with CyQuant was performed utilizing a Fluorstar plate reader (B&L Systems) according to the manufacturer’s instructions.Cells transfected with control or RECQ1 siRNA have been used for radioresistant DNA synthesis assays 48 h immediately after transfection. Cells were labeled for 24 h with 10 nCi/ml [14C]thymidine and then incubated for another 24 h in non-radioactive medium. Thirty min just after irradiation (10 Gy), 2.five mCi/ml [3H]thymidine was added to the medium for 15 min to allow cell labeling. The cells have been collected and transferred to Whatman filters and fixed sequentially with 70% methanol followed by 95% methanol. Radioactivity was measured in a liquid scintillation counter. The measure of DNA synthesis was derived from resulting ratios of [3H]/[14C] and expressed as a percentage of control values. For G2/M checkpoint assays, HeLa cells have been transfected with siRNAs and irradiated 48 h just after transfection. Cells had been harvested 1 h soon after ” irradiation, ethanol-fixed, stained with propidium 856867-55-5 iodide and anti-phosphohistone H3 antibodies followed by Alexa Fluor 488conjugated secondary antibody (Molecular Probes, Carlsbad, CA), and analyzed using a FACScalibur.U2OS cells that were either untreated or exposed to IR (10 Gy) and permitted to recover for 6 h were trypsinized and collected by centrifugation. Cells had been washed once with cold PBS and portioned equally in four eppendorf tubes, and their sub-cellular fractionation was performed as described [19]. Briefly, following centrifugation at 4uC for 3 min at 300 g, one pellet which represented the whole cell pellet (P1) was frozen in liquid nitrogen and the remaining pellets were resuspended in cold buffer A (10 mM PIPES (pH 7.0), 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 300 mM sucrose, 0.five mM Na3VO4, 50 mM NaF, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 1 mM phenylmethylsulfonylfluoride (PMSF)) containing 0.5% Triton X-100 and incubated at room temperature for 2 min to permeabilize cells. Following centrifugation at 4uC for 3 min at 300 g, the supernatant which represented cytosol and nucleosol fractions (S2) was collected and frozen. The pellet was washed with cold buffer A. One pellet which represented detergent-insoluble nuclei (P2) was frozen. The detergent insoluble nuclei from the other pellet have been then digested with RNase-free DNase I (200 U/ml, New England Bio-Labs, Ipswich, MA) in buffer A for 30 min at room temperature followed by centrifugation at 4uC for 3 min at 300 g. The supernatant (S3) was 
8663121 frozen, and the pellet that had been washed with cold buffer A was either frozen (P3) or extracted with cold buffer A containing 0
