These observations indicate that angiogenin is an anti-thrombotic factor

a cell line of the tongue, was cultured in DMEM/F-12 medium supplemented with 10% FBS, 50 g/ml ascorbic acid, 400 ng/ml hydrocortisone and antibiotics. qPCR Total RNA from fresh tissues and cell lines was isolated with the RNeasy mini kit or the mirVana miRNA isolation kit, according to the manufacturer’s protocols. Following DNase I treatment in order to eliminate genomic DNA contamination, 1 g of total RNA per sample was used to generate cDNA using Oligo-dT and reverse transcriptase. The resulting cDNAs were subjected to qPCR using specific primers and SYBR Green PCR master mix in the StepOnePlus Real Time PCR. Gene expression was determined using the delta-delta CT method and the housekeeping gene PPIA was used as reference gene for data normalization. All reactions were performed in triplicate. Pairs of primers are described in S1 Immunohistochemistry Activin A immunostaining was performed using the streptavidin-biotin peroxidase complex method. Briefly, after dewaxing and hydration in graded alcohol solutions, the sections were treated with 3% H2O2 followed by antigen RS 1 manufacturer retrieval with 10 mM citric acid pH 6.0 in a pressure cooker. After washing with phosphate-buffered saline, the sections were treated with 1% bovine serum albumin in PBS for 1 h and then incubated with polyclonal rabbit antibody against activin A, diluted 1:100, followed by the LSAB method. Reactions were developed by incubating the sections with 0.6 mg/ml 3,3′-diaminobenzidine tetrahydrochloride containing 0.01% H2O2. Control reactions were performed by omission of the primary antibody. Activin A expression was assessed with the aid of the Aperio ScanScope CS. Briefly, glass slides were scanned into high-resolution images, which were analyzed in the Pixel Count V9 algorithm software. The tumor cell islands were delimitated and by using specific input parameters, the percentage of cytoplasm positivity was calculated and classified in three range categories, according to its staining intensity as weak, moderate and strong. To each category, an intensity score was set: 1 for weak, 2 for moderate, and 3 for strong staining. Tumor final scores were calculated as the sum of the percentage of each category multiplied by its intensity score, using the following equation:. Treatments Lyophilized recombinant activin A and follistatin were dissolved in culture medium, aliquoted and stored at -80C. To assess the effect of activin A, cells were cultured in 0.1% FBS media containing 0, 1, 10 or 100 ng/ml for 24 h. Follistatin was used at concentration of 100 ng/ml. 4 / 22 Activin A Overexpression in Oral Cancer Stable cells mediating INHBA silence SCC-9 ZsGreen PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19730234 LN-1 cells grown in a 12-well plate at confluence of 50% were incubated with control or INHBA shRNA lentiviral particles in culture media containing 8 g/ml of polybrene for 8 h. After washing with PBS, cells were cultured in fresh media for an additional period of 48 h. Cells were then split in a 1:5 concentration, and cultured for 10 days in the presence of 1 g/ml of puromycin dihydrochloride to select resistant cells. The efficacy of INHBA knockdown was determined by qPCR and enzyme-linked immunosorbent assay. ELISA Conditioned cell culture media was collected and the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19728371 cells harvested using 0.25% trypsin and counted with a cell counter. After centrifugation, microtiter plate wells were coated with 100 l of the conditioned-media for 2 h at room temperature. The wells were then washed 3 times with 400 l of 1% Tween 20 in PBS