At least selleck chemicals llc 100 cells per experimental point were scored for presence of foci, and each experiment repeated at least 3 times independently. Flow cytometric analyses Exponentially growing cells were plated in drug free medium 48 hours before experiment. For proteasome activity assay, GFPu 1 cells were exposed to Inhibitors,Modulators,Libraries drugs at the indicated concentration for 24 hours, then analyzed for green fluorescent protein expression. For cell cycle analyses, cells were exposed to drugs at the indicated concentration for 24 hours, and exposed to Inhibitors,Modulators,Libraries IR 8 hours before being pulse labeled with 30 uM 5 Bromo 2 deoxy Uridine for 15 minutes, washed and fixed with 70% ice cold ethanol. Cells were then stained for DNA content and BrdU incorpo ration with anti BrdU rat monoclonal antibody followed by FITC conjugated goat anti rat antibody.

For HR assays, cells were transfected with pCBASce tagged I Sce1 expression vector) or the empty pCAGGS vector using TransIT transfection reagent following manufacturer recommenda Inhibitors,Modulators,Libraries tions. 24 hours after transfection, cells were treated with the indicated drugs at the indicated concentration Inhibitors,Modulators,Libraries for 24 hours. Cells were then fixed and stained for HA expression with mouse anti HA antibody followed by APC conjugated donkey anti mouse antibody. To specifically deter mine the proportion of HR events in I Sce1 expressing cells, the percentage of GFP positive cells among the HA positive cell population was quantified. Flow cytometric analyses were performed on a Becton Dickinson FACScan. Fluorescence data were plotted using FlowJo. At least three independent experi ments were carried out for each condition.

Proteasome activity fluorogenic assays were per formed as in. Briefly, HeLa cells were treated with the indicated FA pathway inhibitors for 6 hours, scrapped, washed in cold PBS, and lysed by 30 minutes incubation in 5 mM EDTA on ice. Cellular extracts Inhibitors,Modulators,Libraries were cleared by centrifugation and quantified. Fluorogenic peptides specific for the chymotrypsin like, trypsin like and caspase like activities of the proteasome were incubated with 5 ug HeLa extracts in specific substrate buffers. Fluorescence emitted by proteasome cleavage of the peptides was monitored every 200 seconds for 1 hour using a fluorometer with 380 nm and 440 nm excitation and emission filters, respectively, and maximum linear slopes were measured.

Emission of serial dilutions of AMC in extracts was used for fluorometer calibration. Proteasome activity was calculated as concentration of AMC produced per second per mg of protein. Three independent experiments were performed. Drug interaction analysis 2008 and 2008 FANCF cells were plated in 96 well plates at a density of 2000 cells/well. 24 mostly h after plating, cisplatin and FA pathway inhibitors were added concomitantly, or FA pathway inhibitors were added and the cells immediately exposed to IR.