Pression wasAvailable online http://breast-cancer-research.com/content/7/2/RFigureSchematic drawing of the pSilencer1.0_U6 vector The WP1066 cost U6-RNA promoter was cloned in front of the gene-specific targeting sequence (19-nuclevector. otide sequences from the target transcript separated by a short spacer from the reverse complement of the same sequence) and six thymidines (T6) as a termination signal. The predicted secondary structure of the pSilencer -Myc transcript target c-Myc is shown. The transcript, a short hairpin double-stranded RNA (dsRNA), is believed to be further cleaved by Dicer to generate a 21-nucleotide siRNA that forms dsRNA ndonuclease complexes and will bind and destroy c-myc mRNA.quantified with a Gel EDAS analysis system (Cold Spring USA Corporation) and Gel-Pro Analyzer 3.1 software (Media Cybernetics).Cell growth assaykept in pathogen-free environments and checked every 2 days. The date at which a palpable tumor first arose and the weight of the tumor were recorded.Cell cycle analysisAt 2 days after transfection, MCF-7 cells transfected with indicated plasmids were harvested and replated at a density of 50 cells/mm2 in triplicate. The total cell number was quantified every 2 days with a hematocytometer and an Olympus inverted microscope. Cell viability was assessed by using trypan blue.Soft agar colony assayStandard fluorescence-activated cell sorting analysis was used to determine apoptosis of the cells. In brief, MCF-7 cells were transfected with pSilencer -Myc or pSilencer; 24 hours later, cells were deprived of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/29045898 serum for 36 hours. Then cells were harvested, washed once in PBS and stained with propidium iodide (BD Biosciences). The apoptotic cells were assessed by flow cytometric detection of sub-G1 DNA content.TdT-mediated dUTP nick end labelling assayAt 2 days after transfection, MCF-7 cells (300 cells per well) transfected with indicated plasmids were mixed with tissue culture medium containing 0.7 agar to result in a final agar concentration of 0.35 . Then 1 ml samples of this cell suspension were immediately plated in six-well plates coated with 0.6 agar in tissue culture medium (2 ml per well) and cultured at 37 with 5 CO2. After 2 weeks the top layer of the culture was stained with 0.2 piodonitrotetrazolium violet (Sigma). The culture was analyzed in triplicate, and colonies larger than 100 in diameter were counted.Tumor growth in nude miceEqual numbers (106 or 2 ?106) of MCF-7 cells transfected with pSilencer -Myc or pSilencer were harvested by trypsinization 2 days after transfection, washed twice with 1 ?PBS, and resuspended in 0.2 ml of saline. Two groups of five 4?-week-old female nude mice were then given bilateral subcutaneous injections with control cells or cells transfected with plasmids against c-Myc. The mice wereApoptotic cells were confirmed with the in situ cell death detection kit, Alkaline Phosphatase (Roche Applied Science), in accordance with the manufacturer’s instructions. In brief, MCF-7 cells were grown on coverslips. The next day, cells were transfected with pSilencer -Myc or pSilencer. At 24 hours after transfection, cells were deprived of serum for 36 hours. Coverslips with adherent cells were fixed in 4 paraformaldehyde for 1 hour at room temperature and permeabilized with 0.1 Triton X-100 for 2 min on ice. DNA fragments were labeled with the TdT-mediated dUTP nick end labelling (TUNEL) reaction mixture for 60 min at 37 in a humidified atmosphere in the dark. The coverslips w.
