The spectrAlcohol at room temperature for 1 hour. The spectrophotometric absorbance of each sample was then determined using PD173074 ULTRA Multifunctional plate micro Leseger t on at 595 nm. Histone DNA ELISA to detect the cell death by apoptosis Apoptosis was quantified by cell apoptosis detection ELISA kit. PC3 cells were cultured in 6-well plates at a density of 5.0 adhere well sown 104 t and 24 hours, in order to the surface Surface of each well. Then the cells were treated with various concentrations of reagents as described above. After treatment were cytoplasmic histone-DNA fragments of PC cells and extracted 3 on a plate immobilized histone Antique Inserted body. Subsequently End a peroxidase conjugated Antique Body was used connected to the DNA for the detection of DNA fragments histones.
A substrate for peroxidase is then added to each well, containing added each experimental condition. The spectrophotometric absorbance of each sample was then measured using ULTRA Multifunctional Micro-plate A 922500 reader at 405 nm. DNA ladder analysis for detection of apoptosis PC 3 cells were sown in bo t Your 100 mm to 3.5 105 cells dish and you lie they adhere and grow for 36 hours. For growth and attachment PC 3 cells with 15 nM of SB715992 for 48 and 72 hours were treated. After treatment the DNA cytoplasmic cells using 10 mM Tris, 0.5 mM EDTA and 0.2 Triton X extracted 100th The lysate was then centrifuged at 4 for 15 min at 13 800 g to separate DNA fragments of the nuclear cytoplasmic granules. The supernatant was then collected and long with 15 l of RNase and incubated at 37 for 1 hour.
After incubation, the supernatant was 20 with 20 l of SDS, 8 l of proteinase K, 25 l of 5.0 M NaCl and incubated at 37 w Treated during 30 minutes. Subsequently End were phenol-chloroform isoamylalcohol extraction and F Filling conducted with isopropanol. To F Filling, the DNA fragments at 70 and washed with alcohol. On a 1.5 agarose gel at 100 volts for 80 minutes After electrophoresis, the gels were angef Rbt l Runs visualized with ethidium bromide and UV light. The microarray analysis of gene expression profiles of PC3 cells were treated with 10 nM SB715992 for 6, 24 or 48 hours. Total RNA was extracted from each sample with Trizol following manufacturing method, s protocol. The total RNA from each sample was then purified with RNeasy Mini Kit DNase and RNase set following manufacturer’s protocol.
The purified RNA samples were submitted by microarray Human Genome U133A array analysis, which contains 54,613 human gene probes Lt Gene expression was then quantified using Microarray Suite, MicrodB ? and data mining tool. Clustering and annotation of gene expression were analyzed by Cluster and TreeView Onto Express and GenMAPP. Analysis of RNA expression of each reaction Transcriptionpolymerase not turn back to Ver changes In gene expression at the mRNA level, verify that appeared on the microarray, w Hlten we repr Sentative genes with different expression profiles for real-time RT-PCR analysis. PC3 cells were treated with 10 nM SB715992 for 6 to 48 hours, total RNA was isolated and purified as above mentioned Hnt. Two micrograms of total RNA from each sample were subjected to reverse transcription using t