The cells were infected with CNHK600-IL24 and CNHK600-EGFP at MOI of 5. Two hours after incubation with the viruses, the supernatants were discarded and replaced with 3 ml culture medium containing 5% FBS. At timepoints 0, 12, 24, 48, 72 and 96 hours after infection, the cells were scraped and transferred to five-ml centrifuge tubes and underwent three cycles of freezing and thawing between 37°C and −80°C. The TCID50 method was used to determine titre. Cell growth inhibition assay Log phase MDA-MB-231 cells and MRC-5 cells were adjusted
to 1 × 105 cells/ml with culture medium containing selleck inhibitor 10% FBS, and 100 μl/well was added to 96-well plates. The cells were incubated at 37°C for 18 h and then infected with CNHK600-IL24 see more and CNHK600-EGFP at MOI values of 0, 0.1, 0.5, 1, 5, 10, 100 and 1000. Two hours after incubation with virus, the supernatants were discarded and replaced with 100 μl culture medium containing 5% FBS. Five days after infection, 100 μl 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) at 1 mg/ml was added. The plates were incubated at 37°C for 4 h, and then the supernatants were discarded and 100 μl DMSO (Merker) was added. After 15 min shaking,
absorbances at 490 nm were measured. Detection of IL-24 protein in culture supernatants and cells Log phase MDA-MB-231 and MRC-5 cells were adjusted to 1 × 105 cells/ml and added to 6-well plates. The cells were infected with CNHK600-IL24 at a MOI of 5. Two hours after incubation,
the medium was replaced with fresh culture medium supplemented with 5% FBS. Supernatants were collected at 12, 24, 48 and 96 h after infection. mafosfamide The expression of IL-24 was measured with a standard ELISA assay (GBD Biosciences Catalog No. I083). At the same time, cells were lysed on ice with 500 μl lysis buffer (10 mM Tris-Cl, pH 7.4, 0.15 M NaCl, 5 mM EDTA, 1% Triton X100, 5 mM DTT, 0.1 mM PMSF, 5 mM ε-aminocaproic acid) per well. The cell lysates were centrifuged at 10,000 g, 4°C for 10 min, and then the supernatants were stored at −80°C until used for western blotting to detect the expression of IL-24 protein. Establishment and treatment of the orthotopic breast cancer model in nude mice Nu/nu female mice, aged 5- to 6-weeks old and weighing about 18 to 20 g, were cultivated by the Shanghai Experimental Animal Center of Chinese Academy of Sciences. All procedures were approved by the Committee on the Use and Care on Animals and done in accordance with the institution guidelines. Log phase MDA-MB-231-luc cells (Xenogen Corporation) were diluted with Rabusertib research buy sterile PBS to 8 × 107 cells/ml and mixed with matrigel at a 1:1 ratio. After inhalation anesthesia, 50 μl cells were injected into the fat pad of nude mice. At timepoints 14, 16, 18, 20 and 22 days after the injection of cells, viruses were administered through intravenous injection.