Era and genotyping of transgenic mice with cardiac limited overexpression of human Bcl 2 have been previously described. Bcl 2 transgenic mice were on mixed back ground and their non transgenic littermates were used as controls. Doxorubicin therapy was done with intraperitoneal injection of doxorubicin once weekly for 4 weeks. Pitavastatin therapy was conducted with daily oral administration. All animal procedures were done with the approval of the Institutional Animal Care and Use Committee of Chiba University. Transthoracic echocardiography was executed with Vevo 660 equipped with Celecoxib molecular weight a MHz imaging transducer. All recordings were done on conscious animals. Total intracellular oxidation in cultured cardiomyocyteswas evaluated with 2?, 7? dichlorofluorescein fluorescence using CM H2DCFDA. Intracellular oxidative stress was monitored by microscopic observation and measurement of intracellular fluorescence intensity utilizing the Mithras LB940 as previously described. Measurements were carried out for 5 samples in each group based on the manufacturers instruction. Histological detection of superoxide production was considered with DHE as previously described. To Ribonucleic acid (RNA) assess DNA damage in cultured cardiomyocytes, CometAssay was done in line with the manufacturers instruction. Throughout electrophoresis, undamaged DNA remains within the bounds of the nucleus, although broken DNA migrates out of the nucleus in the model of a comet. Each comet was given a of 0 to 4, and 10-0 cells per slide and 3 slides per treatment were analyzed. Paraffin sections of the heart samples fixed in 10 percent formalin were stained with the antibody against phosphorylated histone H2AX and dystrophin, to evaluate DNA damage in the heart in vivo. Western blot analysis was performed as previously described. Whole cell or tissue lysates were employed for analysis, unless mentioned otherwise. For Rac1 subcellular localization assay,membrane and cytosolic proteins were prepared using proteoextract native membrane protein removal package in accordance with themanufacturers training. Particular signals were found using enhanced chemiluminescence. The primary antibodies useful for western blotting were as follows: phospho ATM, ATM, phoshop53, p53, Bax, cleaved caspase 3, Rac1, and actin. NADPH oxidase activity was measured as previously described. All measurements were performed as triplicates in 96 well luminometer dishes. The number of viable cells order AG-1478 in vitro was determined with trypan blue exclusion technique. For apoptosis investigation in vitro and in vivo, TUNEL labeling was performed based on the manufacturers protocol. TUNEL positive cells were counted in 3 randomly selected low power fields from each culture plate, 3 meals for each group in-vitro.