or and normal adjacent tissue was punch-biopsied with a 1-mm tissue microarray needle. Tumor content of the punches was histologically reevaluated to confirm a tumor content.90% in each sample. Frozen matched malignant and nonmalignant samples were collected in RNAlater stabilization reagent. For all patients, the following TG100 115 clinicopathological information was available: Tumor classification according to the International Union Against Cancer 2002 TNM system, tumor grading according to Gleason, follow-up time after surgery and follow-up prostate-specific antigen values. Biochemical relapse was defined as the first PSA value after radical prostatectomy.0.1 and was confirmed by a subsequently elevated value. Additionally, only patients whose PSA levels dropped below detection limit after surgery were considered for analysis. Reagents, cytokines and death receptor ligands For stimulation and apoptosis induction experiments recombinant human TNFa from Active Bioscience was used; human activating antiFas/CD95 was from Upstate; soluble human recombinant TRAIL was purchased from Alexis Biochemicals. For apoptosis inhibition the general caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone from BD-Biosciences was used. miR mimics and inhibitor molecules For transfection experiments synthetic miR-133b, antimiR-133b and their respective scrambled controls: control miR and control antimiR were purchased from Ambion. Unless otherwise specified, negative control and miRs were used at 10-nM concentration. For inhibition experiments amiRs or scrambled 
control were added at 30-nM concentration. All transfections were carried out using Lipofectamine 2000 from Invitrogen according to the manufacturer’s protocol. genes, strongly suggests that downregulation of miR-133b represents an important step during tissue transformation. Hence, further studies should aim at exploring the potential of miR-133b as molecular target for cancer therapy. Apoptosis assays The day before transfection, HeLa or PC3 cells were seeded in 24-well plates at a density of 56104 cells/well. MiR-133b or ctrl miR was transfected either alone or together with ctrl or specific amiR-133b. Forty-eight h after transfection, cells were stimulated for 6 h with the respective proapoptotic stimuli. Apoptotic cells were quantified by flow cytometry by measuring caspase activation status with caspase-specific FLICATM apoptosis detection kits from Immunochemistry Technologies as per manufacturer’s instructions. 7-Amino-actinomycin D was used to exclude cells with damaged cellular membrane from the caspase activation quantification. Propidium iodide incorporation was used for determining overall cell vitality. For this, Materials and Methods Ethics statement The study was approved by the ethical board of the Charite University Hospital and written informed consent has been obtained. Cell lines HeLa and PC3 cells were obtained from the German Collection of Microorganisms and Cell Cultures and analyzed “1981266 by flow cytometry. 133b was normalized to miR-130b. Standard curves were generated for each miRNA to allow for suboptimal efficiencies and to calculate arbitrary concentrations. 3–2,5 diphenyltetrazolium bromide cell survival and proliferation assay MTT was diluted in PBS to a final concentration of 5 mg/ml and sterile filtered. Before use, “2436504 MTT stock solution was further diluted with cell culture medium to a final concentration of 100 mg/ml. To perform the assay, 50 ml of this solution were added to t
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