Up had three parallel wells. After incubation for 14 days at 37 , cells

Up had three parallel wells. After incubation for 14 days at 37 , cells were washed twice with Hank’s solution and stained with hematoxylin solution. The number of colonies containing 50 cells was counted under a microscope. The clone formation efficiency was calculated as (number of colonies/number of cells inoculated) ?100 .Cell migration and invasion assaysAll data were independently repeated at least thrice. SPSS 13.0 and Graph Pad Prism 5.0 software were used for statistical analysis. One-way ANOVA or two-tailed Student’s t-test were applied to determine the differences between group in vitro analyses. The chi-squared test was used to determine the differences of ENO1 protein expression between NSCLC tissues and non-cancerous lung tissues of the lung. A p value of less than 0.05 was considered statistically significant.In vitro cell migration and invasion assays were examined according to our previous study [46]. For cell migration assays, 1 ?105 cells in a 100-l medium without serum were seeded on a fibronectin-coated polycarbonate membrane insert in a transwell apparatus (Corning, USA). In the lower surface, 500 l DMEM with 10 FBS was added as chemoattractant. After the cells were incubated for 10 h at 37 in a 5 CO2 atmosphere, Giemsastained cells adhering to the lower surface were counted under a microscope in five predetermined fields (100?. All assays were independently repeated at least thrice. For cell invasion assays, the procedure was similar to the cell migration assay, except that the transwell membranes were pre-coated with 24 g/ml Matrigel (R D Systems, USA).In vivo tumorigenesis in nude miceAdditional fileAdditional file 1: Figure S1. Stably upregulated ENO1 (A) or downregulated ENO1 (B) did not induce obvious epithelial to mesenchymal morphology transition changes in SPCA-1 or A549 cells. Abbreviations ENO1: -Enolase; MBP-1: c-Myc promoter-binding protein; NSCLC: Non-small cell lung cancer; EMT: Epithelial-mesenchymal transition; qRT-PCR: Quantitative real-time reverse transcription PCR; MTT: 3-(4 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide; LDHA: Lactate dehydrogenase A; FAK: Protein tyrosine kinase 2; PI3K: Phosphoinositide 3-kinase; AKT: V-akt murine thymoma viral oncogene homolog 1; Ang II: Human angiotensin II. Competing interests The authors declare that they have no competing interests. Authors’ contributions QFF, YL, YF, SNH, HYQ, and SWD performed the research; XS, WYF, and ZL designed Nelfinavir (Mesylate) the research study; RLL, YZ, XLY, MYZ, XJD, and YYC performed the statistical analysis; and QFF, RCL, RL, LBL, and WYF wrote the paper. All authors have read and approved the final manuscript. Acknowledgements This work was supported by the Outstanding Young Teacher Training Project of Colleges and Universities in Guangdong Province (No. Yq2013136), New Star Plan of Pearl River Science and Technology from Guangzhou City (No.2011 J2200009), Yangcheng Scholar Research Projects from Universities of Guangzhou (No.12A011D), and Innovation Team Grant of Guangzhou Municipal Education Department (No.13C06). Author details Cancer Center, Traditional Chinese Medicine-Integrated Hospital of Southern Medical University, Guangzhou, Guangdong, People’s PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 Republic China. 2 Cancer Research Institute of Southern Medical University, Guangzhou, Guangdong, People’s Republic China. 3Department of Pathology, Basic School of Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic China. 4Department of Cancer Biotherapy Cen.

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