Anesthesia with ketamine/xylazine mixture. All prostate lobes together with seminal vesicles were harvested. The wet weight of Agomelatine 5-HT receptor inhibitor prostate lobes plus seminal vesicles was recorded after dissecting from the surrounding tissues like the urinary bladder and urethra tract. Prostate wet weight was normalized against animal body weight individually before statistic analysis. All other major organs were inspected for frank toxicity or evidence of metastasis. Harvested tissues were fixed in 4% buffered para formaldehyde overnight and then subjected to paraffin embedding. H&E staining was performed for histological analysis. BrdULabelingAssay, Immunohistochemistry, andWestern BlotAssay For BrdU in vivo labeling, the animals were injected i.p. with 0.5 ml of a 10 mM BrdU solution 2 hr before necropsy/sacrifice. To detect BrdU labeled cells in tissues, paraffin embedded tissue sections were immunostained for incorporated BrdUusingZYMED1BrdU Staining Kit. To detect apoptotic cell death in xenograft tumors, terminal deocynucleotide transferase dUTP nick end labeling assay was conducted using Apo BrdU IHCTM In Situ DNA Fragmentation Assay Kit. Both assays were carried out as described in our previous publication. Immunostaining for anti Ki 67 and anti C/EBPa was conducted as described in our previous publication. Briefly, paraffin embedded tissue sections were de paraffinized and re hydrated. Antigen recovery was achieved by boiling the sections in citrate acid buffer. The sections were incubated with the primary antibodies overnight at 48C and visualized using aZymedTM LAB SA Detection System. For Western blot, after treatment, cells were harvested, rinsed with PBS, and lysed on ice in a radioimmunoprecipitation assay buffer.
Equal amount of proteins from each lysates was loaded onto SDS PAGE gels and immunoblotted with antibodies as indicated in the figures. siRNATransfection and Luciferase Gene ReporterAssay The Silencer1 Select Validated siRNAs against human C/EBPa and C/EBPb genes were purchased from Ambion, Inc. and used according to the manufacturer’s manual. The pE2F TA LUC reporter construct and the internal control reporter pCMV SEAP were described in our previous publications. Cells plated in 6 well tissue culture plates were transfected with the siRNAs for 2 days, followed by another transfection with thereporter constructs using the CytofecteneTM reagent. After treatment, culture supernatants and cell lysates were Idarubicin 57852-57-0 collected for reporter assays. The luciferase reporter activity of each sample was normalized against the corresponding SEAP activity and protein content prior to calculation of the fold induction value relative to the controls. RNAExtraction and Reverse Transcription PCR Total cellular RNAs were prepared using TriZol reagent. To assess mRNA expression, a reverse transcription PCR method was used as described in our previous publication. RT PCR was carried out using a RETROscript Kit as per manufacturer’s instructions. The primers and PCR conditions were described as follows: for human C/EBPa gene, human C/EBPb gene. Human S18 ribozyme RNA was used as the internal control. The primers were synthesized by IDT. The amplification profile was as follows: 958C for 30 sec, 568C for 30 sec, and 728C for 1 min running in a total of 25 cycl.