Expression.Cell viability assayCell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China) enables sensitive colorimetric assays for cell viability. Briefly, GC-7901 cells had been seeded into 96-wellXie et al. Cancer Cell International 2013, 13:18 9 ofplates at 1 ?104 cells per well 24 hrs prior to transfection. Cells have been transfected with klotho expression vector, blank vector, or no vector (PBS) applying lipofectamine 2000 in line with the user manual (Invitrogen, Grand Island, NY, USA). Cells were then continually cultured in growth medium for 72 hrs. Ten l of reagent provided with the kit were added for the cells and incubated for 1 h. Cell viability was assessed applying the microplate reader at 450 nm. All benefits had been normalized to OD values measured from an identically conditioned well with only development medium.Flow cytometry assayPBST and mounted with anti-fade medium. The staining was examined using a fluorescence microscope.Cell treatments with autophagy and apoptosis inhibitorsGC-7901 cells at 70 confluency were transfected with klotho expression vector, blank vector, or PBS as described above. Cells transfected with klotho expression vector or PBS were incubated with ten mM of autophagy inhibitor 3-methyladenine (3-MA) or 20 M of apoptosis inhibitor Z-VAD-FMK for 24 hours. Cells were then harvested for Western blot and/or flow cytometry assay.Statistical analysisGC-7901 cells have been seeded in 10-cm dishes at a density of 2?06 cells per dish. Following cells reached 70 confluency, cells were transfected with klotho expression vector, blank vector, or PBS as described above. Cells had been then trypsinized and suspended with 500 l of binding buffer containing 5 l of Annexin V-FITC and 5 l of Propidium Iodide (Abcam, Cambridge, MA, USA). Right after incubation within the dark for 1 hour, cells were subjected to flow cytometry assay.Construction of klotho gene expression vectorData was analyzed using the SPSS 13.0 (statistical package for the Social Sciences Version 13.0). Two samples have been compared utilizing student t-test. A p 0.05 was viewed as statistically important.Abbreviations GC: Gastric cancer; PTEN: Phosphatase and tensin homolog; IGF-1: Insulin/ insulin-like growth factor-1; IRS-1: Insulin receptor substrate 1; PI3K: Phosphoinositide 3-kinase; LC3: Microtubule-associated protein light chain three; Akt: Protein Kinase B; mTOR: Mammalian target of rapamycin; 5-Aza: 20-deoxy-5-azacytidine; 3-MA: 3-methyladenine. Ferric maltol web Competing interests All authors declared no conflict of interest. Authors’ contributions BX: Experiment design, acquisition of information, analysis and interpretation of data, preparation of manuscript. JZ: acquisition of data. GS: acquisition of data. DL: Methyl anisate In stock conception and design, revising manuscript critically for crucial intellectual content. JZ: evaluation and interpretation of data. JC: conception and design and style. LY: conception and design and style, final approval of manuscript. All authors study and authorized the final manuscript. Author details 1 Departemt of Geriatric Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China. 2Department of Common Surgery, 8th Changsha Hospital, Changsha, Hunan 410015, China. Received: 20 January 2013 Accepted: 13 February 2013 Published: 21 February 2013 References 1. Kim K, Chun KH, Suh PG, Kim IH: Alterations in cell proliferation connected gene expressions in gastric cancer. Crit Rev Eukaryot Gene Expr 2011, 21:237?54. two. Jang BG, Kim WH: Molecular pathology of g.