Flow cytometric analyses Exponentially growing cells were plated in drug cost-free medium 48 hours before experiment. For proteasome activity assay, GFPu 1 cells had been exposed to drugs at the indicated concentration for 24 hours, then analyzed for green fluorescent protein expression. For cell cycle analyses, cells had been exposed to drugs in the indicated concentration for 24 hours, and exposed to IR eight hours prior to getting pulse labeled with 30 uM five Bromo two deoxy Uridine for 15 minutes, washed and fixed with 70% ice cold ethanol. Cells have been 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 had been transfected with pCBASce tagged I Sce1 expression vector or the empty pCAGGS vector applying TransIT transfection reagent following manufacturer recommenda tions.
24 hours soon after transfection, cells have been treated using the indicated drugs at the indicated concentration for 24 hours. Cells have been then fixed and stained for HA expression with mouse anti HA antibody followed by APC conjugated donkey anti mouse antibody. To especially deter mine the proportion of HR events in I Sce1 expressing cells, the percentage of GFP supplier NVP-TAE226 good cells amongst the HA constructive cell population was quantified. Flow cytometric analyses were performed on a Becton Dickinson FACScan. Fluorescence information had been plotted employing FlowJo. At the least 3 independent experi ments have been carried out for each condition. Proteasome activity fluorogenic assays had been per formed as in.
Briefly, HeLa cells were treated using the indicated FA pathway inhibitors for 6 hours, scrapped, washed in cold PBS, and lysed by 30 minutes incubation in five mM EDTA on ice. Cellular extracts had been cleared by centrifugation and quantified. Fluorogenic peptides distinct for the chymotrypsin purchase NVP-BEZ235 like, trypsin like and caspase like activities on the proteasome were incubated with 5 ug HeLa extracts in specific substrate buffers. Fluorescence emitted by proteasome cleavage of your peptides was monitored each and every 200 seconds for 1 hour employing a fluorometer with 380 nm and 440 nm excitation and emission filters, respectively, and maximum linear slopes have been measured. Emission of serial dilutions of AMC in extracts was utilised for fluorometer calibration. Proteasome activity was calculated as concentration of AMC made per second per mg of protein.
3 independent experiments have been performed. Drug interaction analysis 2008 and 2008 FANCF cells have been plated in 96 well plates at a density of 2000 cells well. 24 h immediately after plating, cisplatin and FA pathway inhibitors have been added concomitantly, or FA pathway inhibitors were added along with the cells immediately exposed to IR. Cytotoxicity was measured employing the stand ard crystal violet assay 6 days right after drug addition, cells have been washed twice with PBS, fixed for 5 minutes at area temperature in 10% methanol and 10% acetic acid.