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  • br Materials and methods br Antibodies

    2019-10-21


    2. Materials and methods
    2.1. Antibodies and reagents
    The antibody against cytochrome c (#sc-13156) and caspase-3 were (#sc-7148) purchased from Santa Cruz Biotechnology, Inc. The anti-body against GAPDH was from Good HERE Biotech Inc. (Hangzhou, China, #AB-P-R001), and anti-COX IV antibody was from Abbkine Inc. (CA, USA, #ABM40033). The primary Suramin hexasodium salt for VDAC (#55259-1-AP) and CypD (#12716-1-AP) were purchased from Proteintech Group, Inc. (Wuhan, China). The horseradish peroxidase-conjugated secondary antibodies were obtained from Zhongshan Golden Bridge Biotech Inc. (Beijing, China, #ZB-2301). FITC-Annexin V/ PI apoptosis assays kit was from Biovision, Inc. (Palo Alto, CA). The JC-1 probe and cell mi-tochondria isolation kit were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Two PTPC inhibitors, DIDS and CsA, were respectively from Sigma (St. Louis, USA) and J&K Scientific Ltd. (Beijing, China). Enhanced chemiluminescence (ECL) detection re-agents were from Pierce Biotechnology, Inc. (Rockford, IL). Propidium iodide (PI) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) were from Sigma (St. Louis, USA).
    DN3 was synthesized based on the scaffold of Jaridonin, a nature 
    ent-kaurane diterpenoid, in our laboratory. The synthetic procedures were shown in Supplementary Fig. S1 and chemical structure was confirmed by IR and NMR data (Fig. S2, S3 and S4). Purity was de-termined by HPLC and was above 99.9%. DN3 was dissolved in di-methyl sulfoxide (DMSO), aliquoted, and stored at − 80 °C. The con-centration of DMSO in culture medium was kept below 0.1% (v/v), a concentration known not to affect cell proliferation (Zi, Simoneau, 2005).
    2.3. Cell culture conditions
    Human gastric cancer cell lines MGC-803 and HGC-27 were pur-chased from China Center for Type Culture Collection (CCTCC, Shanghai, China) and cultured in RPMI 1640 medium. The human immortalized gastric mucosa epithelial cell line GES-1was preserved in our institute and considered as a normal cell line. The GES-1 cells were derived from a human fetal gastric mucosa epithelium, which main-tained normal cytoskeleton and were non-tumorigenic in nude mice (Ke et al., 1994; Jiang et al., 2017). The cells were cultured in DMEM medium. All media were supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS). All the cell lines were incubated at 37 °C in a humidified atmosphere, containing 5% CO2.
    2.4. Cell viability assay
    MGC-803 and HGC-27 cells were plated at a density of 6 × 103 cells/well in 96-well plates in complete culture medium. The GES-1 cells were seeded in 96-well plates at 1 × 104 cells/well, which could maintain untreated GES-1 cells in an exponential phase of growth during the entire experiment. The following day, the cells were re-freshed with fresh medium, untreated or treated as indicated in the figure legends. 24 h or 48 h later, MTT was added at a final con-centration of 1 mg/ml and incubated at 37 °C for 4 h. The absorbance was determined at 490 nm. All experiments were repeated at least three times. The inhibition curves and IC50 values were calculated with GraphPad Prism vision 6.01.
    2.5. Apoptosis analysis by flow cytometry
    As we have described previously (Ma et al., 2013), FITC-annexin V and PI was used to stain and identify Suramin hexasodium salt subpopulations of cells with membrane changes (early stage apoptosis) and the associated loss of membrane integrity (necrotic or late apoptotic cells), respectively. Briefly, cells were treated with the indicated concentrations for 24 h, centrifuged and washed with binding buffer (Biovision). Following the apoptosis detection kit's protocol (Biovision), cells were incubated with 200 μl of binding buffer containing FITC-Annexin V and propidiumio-dide for 15 min at room temperature in the dark. Samples were kept in a light-resistant container at 4 °C before flow cytometry analysis. For each sample, 10,000 cells were analyzed by Accuri C6 flow cytometry (Becton, Dickinson & Co.; Franklin Lakes, NJ). Data were processed using FlowJo Version 7.6.