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  • br VEGF Luciferase Reporter Assay br

    2020-03-17


    VEGF-Luciferase Reporter Assay
    The hVEGF-pGL2 promoter reporter was provided by Dr. Barbara K. Vonderhaar (Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD). MDA-MB-231 or MDA-MB-468 cells were cotransfected with hVEGF-pGL2 with Renilla luciferase Reporter (as internal control) and then maintained in medium with 0.5% FBS and various con-centrations of wogonoside (0, 25, 50 and 100 mM) for 24 h. The luciferase activity of cell lysate was determined by the Dual-Luciferase Reporter kit. Luciferase signals were collected by DualLuciferase assay system (Thermo Fisher Scientific, Rockford, IL).
    Western Blot Analysis
    After treatment with wogonoside (0, 25, 50 and 100 mM) for 24 h, cell extracts were obtained by lysis with RIPA buffer. Protein samples were separated by SDS-PAGE and transfered to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). Membranes were incubated with the primary DETA NONOate (Key Resources Table) specific for target proteins overnight at 4 C, and then incubated with the secondary antibody which was coupled to infrared (IR) dyes for one hour at room temperature. Detection was performed by the Odyssey Infrared Imaging System (LI-COR Inc., USA) using a fluorescent readout and quantified using the Image Studio 5.2. software (LI-COR, USA).
    Real-Time PCR Analysis
    Total RNA was extracted after treated with wogonoside (0, 25, 50 and 100 mM, 24 h) using Total RNA Extraction Reagent (Vazyme). cDNA was made using 500 ng of total RNA with Hiscript II Reverse Transcriptase (Vazyme). qPCR incubations were run with 200 nM of gene-specific primers and HiScript II Q RT SuperMix for qPCR (Vazyme). The qPCR primer sequences and protocol were described in Key Resources Table. qPCR data was analyzed by the DDCt method.
    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
    Gene Knockdown
    The target siRNA sequences against Cul4A (siCul4A-1, 5’- GCACAGAUCCUUCCGUUUA-3’; siCul4A-2. 5’- GGUUUAUCCACGGU AAAGA-3’), and the scrambled siRNA (siControl) were synthesized by Gima Company. Cells were transfected with 25 nmol/l siCul4A using lipofectamine 2000 (invitrogen). The siRNA-transfected MDA-MB-231 cells were used for subsequent experiments after 24 hours. Knockdown efficiency of Cul4A was verified by RT-PCR.
    Molecular Docking Studies
    Molecular docking simulations were used to explore the potential interaction of wogonoside on Gli1 or SMO. The crystal structure of Gli1 (PDB: 5ED2) and SMO (PDB: 4N4W) were prepared by the Protonate 3D tool in MOE (version 2010.10, Chemical Computing Group Inc. Montreal, Quebec, Canada, 2010) and all the water molecules were removed. Hydrogen atoms were added using MOE. The structure of wogonoside was modeled and minimized in MOE. Docking simulations were carried out in the CDOCKER module implemented in Discovery Studio 4.0 (version 2.5, Accelrys Inc., San Diego, CA, 2009).
    BODIPY-cyclopamine Based Fluorescence Binding Assay
    293 cells were transfected with a human SMO expression vector in 6-well plates. Transfected cells were washed with phosphate-buffered saline (PBS) cantaining 1% fetal bovine serum, fixed in 4% paraformaldehyde in PBS and treated with 10 nM BODIPY-cyclopamine and different concentrations of the wogonoside at 37 C for 3 h. Cell nuclei was also stained with DAPI. The fluorescence images were captured and analyzed with Cytation 5 (Biotek, Winooski, VT).
    Cellular Thermal Shift Assay (CETSA)
    Cellular thermal shift assay was conducted as described previously (Wu et al., 2018). In briefly, MDA-MB-231 cells were treated with DMSO or 100 mM wogonoside for 12 h, and then cells were collected and resuspended in phosphate buffered saline (PBS). Each aliquot (33106 cells) were heated at indicated temperatures for 3 min on PCR instrument (Bio-Rad, CA) and frozen twice in liquid nitrogen. The samples were centrifuged and the supernatants were subjected to Western Blotting detection.