Clonogenic assay. Single cells were plated, treated with PHA665752, and 24 hours later exposed to IR using a 137Cs irradiator. One day after IR, PHA665752 was removed. Ten days after plating, cells were fixed and stained with 2 crystal violet. Clonogenic survival was determined using Colcount, Charm Enhanced Algorithmus. SGX-523 Colonies of 50 cells were scored. Clonogenic fraction of irradiated cells was normalized to plating efficiency of nonirradiated controls. Antibodies. Rabbit anticleaved caspase 3, anticleaved lamin A, and antiphospho MET, ATR, CHK1, and CDC25B antibodies were all purchased from Cell Signaling Technology . Mouse antiphospho histone H2A.
X and antiphospho ATM were obtained from Upstate Biotechnology Inc Rabbit anti MET and mouse anti JNK1 antibodies were from Santa Cruz Biotechnology, mouse antiphosphotyrosine PY 20 from BD Biosciences, Hesperadin and rabbit anti actin antibody from Sigma . Western blotting and immunoprecipitation. Cells were lysed, and protein concentration was determined as described previously.41 Proteins were resolved by SDSPAGE, transferred onto PVDF membranes, and incubated with antibodies. Secondary antibodies conjugated to horseradish peroxidase were detected by an ECL kit. ECL signals were quantified using Quantity One software. For immunoprecipitations, lysates were incubated with 1 g of antibodies, and subsequently, MACS protein G Microbeads were added. After calibration, columns were loaded with samples and washed with high salt and low salt buffers.
Beads were boiled with sample buffer and immunoprecipitated complexes analyzed by SDS PAGE. Caspase 3 assay. Caspase 3 activity was assessed via a fluorogenic assay utilizing the Ac DEVD AMC specific caspase 3 substrate. Cells were lysed and analyzed for caspase 3 activity in assay buffer. After substrate addition, fluorescence was measured with a TECAN Infinite200 plate reader. Caspase 3 activity was normalized to samples, protein content. Confocal microscopy. Cells were prepared as described previously,15 incubated with anti ?H2AX antibody, labeled with secondary goat antimouse cyanine 2 antibody , and mounted in PBS:glycerol containing 170 mg mL Mowiol 4 88. For analysis, a Zeiss LSM 510 Meta was used. Images were processed using IMARIS software. Positive ?H2AX foci per cell were counted. Synergism calculations.
CIs were calculated according to Chou and Talalay.21 Briefly, if for a given combination, dosages D C1 and D C2 of drugs 1 and 2, the fraction of affected cells is f A and the drugs are mutually exclusive, CI is defined as CI D S1 D C1 D S2 D C2 , where D S1 and D S2 are the separate dosages of drugs 1 and 2, respectively, needed to achieve the same affected fraction f A . In our case, values f A , D C1 , and D C2 were directly measured, while D S1 and D S2 were inferred from individual dose effect relations of the used drugs. These relations have the form f A f U m, where f A and f U are the affected and unaffected fraction, respectively, D is the dosage of an individual drug, and D m and m are parameters characterizing the drug,s effect, they can be estimated by measuring f A for several different dosages D. Finally, for a particular combination of dosages of 2 drugs or of 1 drug and IR, CI 1 indicates
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