PMC:5337621 / 30867-35524 JSONTXT

{"project":"2_test","denotations":[{"id":"28218735-24739462-79088353","span":{"begin":1275,"end":1277},"obj":"24739462"},{"id":"28218735-26782714-79088354","span":{"begin":2890,"end":2891},"obj":"26782714"},{"id":"28218735-17406544-79088355","span":{"begin":2970,"end":2972},"obj":"17406544"},{"id":"28218735-17098936-79088356","span":{"begin":3558,"end":3560},"obj":"17098936"},{"id":"28218735-15991339-79088357","span":{"begin":4116,"end":4118},"obj":"15991339"},{"id":"28218735-21886100-79088358","span":{"begin":4120,"end":4122},"obj":"21886100"}],"text":"Materials and Methods\n\nCell culture, plasmids and transfections\nGastric cell lines AGS and BGC823 were cultured in RPMI 1640 (Gibco, Carlsbad, CA, USA) HEK293T and mouse embryonic fibroblast (MEF) were cultured in Dulbecco's modified eagle medium (DMEM) (Gibco). Cell culture media were supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 U/ml ampicillin and 100 μg/ml streptomycin (Gibco). Treatments with doxorubicin (Sigma-Aldrich, St Louis, MO, USA), doxycycline (Clonetech, Shiga, Japan), VX-680 (Selleck Chemicals, Kava Technology), cycloheximide (Amresco, Solon, OH, USA) and MG132 (Sigma-Aldrich) were carried out as indicated. Cells were transfected using lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). For details, see Supplementary Materials and Methods.\n\nPatients and clinical tissue specimens\nSee Supplementary Materials and Methods\n\nImmunoblotting and immunoprecipitation\nCells, gastric cancer tissues and paired normal adjacent tissues were lysed on ice with RIPA buffer. For further details, see Supplementary Materials and Methods. Detailed information about the antibodies used in this study is listed in Supplementary Table 4.\n\nImmunofluorescence staining\nImmunofluorescence staining was performed as described.55 For details, see Supplementary Materials and Methods.\n\nImmunohistochemical staining\nBoth staining intensity and extent were included to evaluate AURKA or Survivin expression. Evaluation was done by at least two independent pathologists. Moderate or strong cytoplasm staining was considered as positive reaction. In analysis, specimen was determined as high staining when \u003e30% cells showed visible brown granules.\n\nCell survival (MTT) assay\nCells were seeded into 96-well flat bottom plates and exposed to increasing doses of VX-680 (Kava Technology, San Diego, CA, USA), doxorubicin (Sigma-Aldrich) separately or combination. Standard 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed.\n\nTUNEL assay\nCells were seeded in a 6-well plate, collected and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min. Cells were then labeled by TdT-mediated dUTP Nick-End Labeling Kit (BD Biosciences, San Jose, CA, USA) and analyzed on a Beckon Dickinson FACScan.\n\nEvaluation of drug interactions\nThe interaction between VX-680 and doxorubicin was analyzed to determine whether the combination was additive or synergistic. This program is based on the Jin's method, which is performed based on the following equation: q=D1+2/(D1+D2−D1 × D2), where D1+2 indicates the effect when cells were used in combination with drug 1 and 2, and D1 or D2 indicates the effect when used alone.\n\nGST pulldown for identifying interacting transcription factors with AURKA\nTo identify transcription factors that interact with AURKA, 500 μg AGS cells nuclear extract, prepared as described previously.8 In-gel digestion and recovery of peptides were performed as described earlier.56 For details, see Supplementary Materials and Methods.\n\nIn vitro ubiquitylation assay\nThe in vitro ubiquitylation assay was performed in a volume of 50 μl containing 50 mm Tris pH 7.6, 0.5 m DTT, 5 mm MgCl2, 2 mm ATP, 50 nm E11, 0.5 μm UbcH5B, 0.5 μm UbcH5C, 0.5 μm UbcH7, 2 μm ubiquitin-Flag, 20 nm Rbx1, 20 nm Cul1, 20 nm Skp1, 20 nm FBXL7, 200 nm Survivin at room temperature for 1 h. Reaction was stopped by addition of SDS–PAGE loading buffer and Survivin ubiquitylation visualized by immunoblotting with Flag antibody.\n\nChIP assay\nChIP assay was performed as previously described.57 For details, see Supplementary Materials and Methods.\n\nRNA extraction and RT–qPCR assays\nTotal RNA was prepared using TRIzol (Invitrogen), according to manufacturer's instructions. For details, see Supplementary Materials and Methods. Primers were purchased from Invitrogen and primer sequences are listed in Supplementary Table 4.\n\nIn silico protein–protein interaction\nTertiary-structure-based in silico protein–protein interaction between AURKA and FOXP1 was determined by PRISM method which is based on template matching with known protein structures.29, 30 We obtained PDB files; 2 × 81 (AURKA) and 2KIU (FOXP1) as a target set of proteins and analyzed their predicted protein–protein interaction as described elsewhere.58\n\nPromoter assay\nPromoter dual luciferase assays (Promega, Madison, WI, USA) were performed as per manufacturer's instructions. For details, see Supplementary Materials and Methods.\n\nStatistical analysis\nStatistical analysis was performed using SPSS v. 16.0 (SPSS, Chicago, IL, USA). All P-values quoted were two-sided. P\u003c0.05 was considered statistically significant. "}