William S. Dalton. All MM cell lines were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U ml penicillin and 100 ug ml streptomycin, and maintained at 37 C in a humidified atmosphere in the presence of 5% CO2 95% air. Constructs and transfection The constructs of APE1 knockdown Perifosine and wildtype or over expression mutants used in this study included pTer, kind gifts from Dr. Gianluca Tell. The APE1 eukaryotic overexpression vector was constructed based on the pcDNA 3. 1 vector. The detailed procedures were reported previously. The transfections were performed using Lipofectamine 2000 Transfection Reagent following the manufacturers protocol for transient transfection of suspension cells. CCK 8 assay Cells on 6 well plates were transfected or treated as indicated.

Cell viability was evaluated by MTT assay at various time points after transfection or treat ment. Cell counting kit 8 reagent was added to each dish at a concentration of 1 10 volume, and the plates were incubated at 37 C for an additional 4 h. Absorb ance was then measured at 490 nm and at 630 Inhibitors,Modulators,Libraries nm as a reference with a Microplate Reader 550. Cell viability OD value of treatment group OD value of control group 100%. Western blot and antibodies Western blots were performed as previously described. Suppliers and incubation conditions of antibodies used for Western blots were as follows, anti APE1 mono clonal, 1 h at 37 C, dilution 1,5000, anti MDR1 monoclonal, dilution 1,500, HRP conjugated anti acetylation lysine antibody, Inhibitors,Modulators,Libraries dilution 1,1000, overnight at 4 C, anti B actin monoclonal, 1 h at 37 C, dilution 1,2000.

Quantitative RT PCR Expression of the APE1 gene was detected by real time RT PCR and normalized by control gene B actin expres sion. Total RNA was extracted Inhibitors,Modulators,Libraries using the TRIZOL re agent and then reverse transcribed into single stranded DNA using PrimeScript 1st Strand cDNA Synthesis Kit. Real time RT PCR was per formed with a Lightcycler 480 real Inhibitors,Modulators,Libraries time RT PCR system. APE1 forward primer, were utilized. Oligonucleotide cleavage assay The AP endonuclease activity of APE1 was evaluated by a well characterized oligonucleotide cleavage assay. Briefly, a 51 mer oligonucleotide containing a THF site, the analogue of an abasic site, at the 22nd position was 5 end radiolabeled. The labeling reaction consisted of 10 pmol of the single stranded oligonucleotide, 2.

Inhibitors,Modulators,Libraries 5 pmol of 32P ATP, T4 PNK, and appropriate kinase buffer in a total volume of 10 ul. Reactants were incubated for 30 minutes at 37 C and 5 minutes at 95 C. Complemen tary oligonucleotide was then added and cooled down to 22 C to form duplex DNA. Activity assays gefitinib lung contained 0. 5 pmol of labeled duplex oligonucleotide, 1 REC Buffer, protein extraction in a 10 ul reac tion volume and were incubated at 37 C for 15minutes. The reactions were terminated by adding 10 ul formam ide with dyes.