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  • br dE i br Herein we performed a

    2020-03-17


    dE ðiÞ
    Herein, we performed a breast-specific co-expressed protein-protein interactome network analysis by comparing genome-wide Conessine profiles of breast to 31 other tissues from GTEx database (GTEx Consortium, 2015). Specifically, we assembled
    Please cite this article in press as: Huang et al., A Systems Pharmacology Approach Uncovers Wogonoside as an Angiogenesis Inhibitor of Triple-Negative Breast Cancer by Targeting Hedgehog Signaling, Cell Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.05.004
    15 commonly used databases with various types of experimental evidence and an unbiased, systematic human protein-protein interactions (PPIs) we have recently utilized (Cheng et al., 2018): (i) binary PPIs tested by high-throughput yeast-two-hybrid (Y2H) systems in which we combined binary PPIs from two publicly available high-quality Y2H datasets (Rolland et al., 2014; Rual et al., 2005); (ii) kinase-substrate interactions; (iii) signaling networks from SignaLink2.0 (Fazekas et al., 2013); (iv) binary PPIs from three-dimensional (3D) protein structures reported in Instruct (Meyer et al., 2013); and (v) carefully literature-curated PPIs identified by low-throughput experiments. All inferred data (e.g., evolutionary analysis and gene co-expression data) are excluded. The detailed descriptions for building human protein-protein interactome are provided in our previous studies (Cheng et al., 2014, 2018; Cheng et al., 2015).
    We next reconstructed the breast-specific co-expressed PPI network by mapping RNA sequencing data from TCGA (Cancer Genome Atlas Network, 2012). Normalized RNA sequencing data (RPKM) were extracted using the R package from TCGA-Assem-bler (Zhu et al., 2014). We computed the co-expression (Pearson correlation coefficient) and p-value (F-statistics) for each PPI based on RNA sequencing data in TNBC. Finally, we only used the p-value (P) < 0.05 as co-expressed PPIs to build the TNBC-specific co-expressed human protein-protein interactome network for network analysis in Figure 2A.
    Compound Preparation
    Wogonoside (>98% purity; Langze Pharmaceutical Co, Ltd., Nanjing, China) was dissolved in dimethylsulfoxide (DMSO) as stock solution at 0.1 M and stored at -20 C. Wogonoside stock solution was freshly diluted with medium to the final concentration before each experiment in vitro. The final DMSO concentration did not exceed 0.1%. In the in vivo study, wogonoside was prepared as intra-gastric administration (0.5% Sodium Carboxymethyl Cellulose, CMC). Chemicals and reagents used in this study were described in Key Resources Table.
    Immunohistochemistry
    Tumor xenografts of mice in control and wogonoside (80 mg/kg) treated groups were immersed in 4% formaldehyde (pH 7.4) for 24 h, embedded in paraffin, cut into sections 4 mm thick using standard histological techniques to prepare paraffin sections. The expres-sion of CD31, VEGF, SMO and Gli1of the tissues from control and wogonoside (80 mg/kg) treated groups were assessed using Immu-nohistochemistry Application Solutions Kit with specific antibodies (1:100).
    Cell Viability Assay
    The MTT assay was used to evaluate the viability of human breast cancer cells. MDA-MB-231 and MDA-MB-468 cells were plated at a density of 2 3 105 cells per well into 96-well plates in medium with 10% FBS. After overnight growth, cells were treated with various concentrations of wogonoside (0, 25, 50 and 100 mM) for 24 h in 5% CO2 incubator at 37 C. Then 20 ml of 0.5% MTT were added into the medium and incubated for 4 h. The supernatant was removed and 100 ml DMSO was added to dissolve the precipitate. Absor-bance was measured at 570 nm.
    Enzyme-Linked ImmunoSorbent Assay (ELISA)
    The concentration of human VEGF protein in MDA-MB-231 CM or MDA-MB-468 CM was determined by a human VEGF Duo-set enzyme linked immune sorbent assay kit in accordance with the manufacture instructions.