The sections were incubated with the TUNEL reaction solution for 60 min at 37uC in the dark

added. Western blot analysis Proteins were extracted, using a RIPA buffer containing protease inhibitors as previously described. Samples were electrophorised on denaturing 15% SDS-polyacrylamide gels as described above. Gels were allowed to equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20% methanol. Proteins were transferred to a 0.2 mm pore size nitrocellulose membrane at 250 mA for 3 h, using a Mini Trans-Blot electrophoresis cell according to the method described in the manual accompanying the unit. The nitrocellulose membrane was blocked with 5% non-fat dry milk for 2 h at room temperature and incubated with caspase 8 or caspase 9 antibodies for overnight at 4uC. Three consecutive washes in PBS containing 0.1% Tween were performed and the membrane was incubated with a secondary antibody conjugated to Horse Radish Peroxidase for 2 h at room temperature. The nitrocellulose paper was washed three times 2181489 and finally, was reacted with enhanced chemiluminescent substrate for 5 min, dried with Whatman sheet and exposed to X-ray film. Cell Viability MCF-7 and T47D cells were seeded in 96-well plates. The following day, cells were treated with BP-C1 in a spectrum of concentrations ranging from 100 to 1,000 mg/ml for 48 hours and cell viability was detected using Cell Proliferation Assay, XTT. This assay is based on the ability of metabolic active living cells to reduce the tetrazolium salt, XTT, to orange colored compounds of formazan. The SB203580 chemical information absorbance of each sample was measured using an ELISA reader at a wavelength of 450 nm with a reference absorbance of 620 nm. The results are presented as percentage of control and expressed as means 25090924 6 standard deviation of three independent experiments in which each treatment was performed in triplicates. Lactate dehydrogenase release Cellular damage, such as necrosis, causes an elevation of the LDH concentration in the medium. The integrity of the plasma membrane following treatment was determined by measuring LDH activity released into the culture medium. The enzyme activity was measured using a spectrophotometric method. Gene expression analysis Gene expression was detected using the Applied BiosystemsH TaqManH Apotosis Array Plate for human apoptosis. The panel of genes in this plate contains 92 Taqman assays for apoptosis associated genes and 4 Taqman assays of endogenous control genes. This panel targets genes from both, the signaling pathways that initiate mammalian apoptosis, the death receptor regulated pathway, and Cell Cycle Analysis Cells were trypsinized, fixed in 70% ethanol and stored at 4uC until a FACS analysis was conducted. The ethanol was removed BP-C1 Induced Apoptosis in Breast Cancer Cells the BCL-2 family pathway, together with few caspases involved in the final mechanisms of cell death. After treatment, total RNA was isolated using the TRIzol reagent. 1 mg of RNA was reverse transcribed using a reverse transcription system according to the manufacturer’s instruction. Then, quantitative real-time PCR amplification was performed using Taqman array plate, to which the synthesized cDNA was added. Each well contains sequence specific primers for known gene and dye-labeled MGB probe, that enables detection of amount of cDNA real time amplification of each targeted gene in the plate. Housekeeping genes, HPRT1 and GAPDH, were used for internal control to correct the potential variation in RNA loading. All reactions were performed on the STEPONEPLUS Real-Time PCR system and i