The Titer TACS colorimetric apoptosis detection kit was obtained from Trevigen, Inc.. The Quantikine M human cytochrome c assay kit and caspase 3 assay kit were purchased from R&D systems. Antibodies have been bought from Santa Cruz Biotechnology, Inc.. Carboplatin, Akt inhibitor, horseradish peroxidase conjugated antimouse IgG, z Asp Gln Met Asp fluoromethyl ketone and z Ile Glu Thr Asp fluoromethyl ketone had been bought Afatinib molecular weight from EMD Calbiochem. Co.. SuperSignal West Pico chemiluminescence substrate for cytochrome c detection in western blot was obtained from PIERCE Biotechnology Inc.. 3 2,5diphenyltetrazolium bromide, monoclonal anti p21 Bax, z LeuGlu His Asp fluoromethyl ketone and other chemicals were bought from Sigma Aldrich Inc.. 2. 2. Cell culture NIH OVCAR three and SK OV 3 cell lines were obtained from Korean cell line bank, and had been cultured in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum, 100 U/ml of penicillin and 100 ug/ml of streptomycin.

Cells had been washed with RPMI 1640 medium Plastid containing 1% fetal bovine serum 24 h before experiments and seeded onto 96 and 24 well plates. 2. three. Cell viability assay Cell viability was measured using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases. The MTT assay provides the rapid and precise results for cellular growth and survival. Cells had been incubated in the absence or presence of Akt inhibitor and carboplatin for 24 h at 37 C. The medium was incubated with 10 ul of 10 mg/ml MTT solution for 2 h at 37 C. After centrifugation at 412?g for 10 min, culture mediumwas removed and 100 ul dimethyl sulfoxide was added to each well to dissolve the formazan. Absorbance was measured at 570 nm using a microplate reader.

Cell viability was expressed as a percentage of the value in control cultures. 2. 4. Morphological observation of nuclear change OVCAR three cells had been incubated in Docetaxel structure the absence or presence of Akt inhibitor and carboplatin for 24 h at 37 C. Then the nuclear morphological change was assessed using the Hoechst dye 33258. Cells were incubated with 1 ug/ml Hoechst 33258 for three min at room temperature and nuclei have been visualized using an Olympus Microscope with a WU excitation filter. 2. 5. Measurement of oligonucleosomal DNA fragmentation The DNA fragmentation due to activation of endonucleases was assessed by gel electrophoresis. Cells had been incubated in the absence or presence of Akt inhibitor and carboplatin for 24 h at 37 C, and then have been washed with phosphate buffered saline.

DNA was isolated with the DNA purification kit, according to the manufacturers directions. DNA pellets have been loaded onto a 1. 5% agarose gel in Tris acetate buffer and 1 mM EDTA, and separated using 100 V for 2 h. DNA fragments had been visualized using a UV transilluminator after staining with ethidium bromide.