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  • br Materials and methods br Cell cultures and

    2020-08-02


    Materials and methods
    Cell cultures and reagents
    Human TNBC cell lines (MDA-MB-231, BT-549, HCC70, and HCC1806) and non-tumorigenic breast cell line (MCF-10A) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). A normal breast cell line (AG11132) was obtained from Coriell Institute for Medical Research (Camden, NJ, USA). TNBC cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium while non-tumorigenic breast cells (MCF-10A and AG11132) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or Mammary Epithelial Cell Growth Medium (MEGM), respectively. The media contained 10% fetal calf serum (FCS) and were cultured in a humidified Nutlin-3 with 5% CO2 at 37 C. Cells Nutlin-3 between the 3rd and 10th passages were used for this study. Nor-wogonin was purchased from Chem Faces (Wuhan, Hubei, China) whereas wogonin and wogonoside were purchased from Sigma-Aldrich (St. Louis, MO, USA).
    Proliferation and viability assays
    Cell proliferation was quantified in terms of bromodeoxyur-idine (BrdU) incorporation using a colorimetric cell proliferation BrdU ELISA kit (Roche Diagnostics, Indianapolis, IN, USA), according to the manufacturer’s instructions. TNBC cells (MDA-MB-231, BT-549, HCC70, and HCC1806) and non-tumorigenic breast cells (MCF-10A and AG11132) were seeded at 5 103 cells/ well and cultured overnight in a 96-well plate. The cells were treated with nor-wogonin, wogonin, wogonoside, or dimethyl sulfoxide (DMSO; vehicle) for 24 h. BrdU was added at a final concentration of 10 mM and cells were cultured for 2 h. BrdU incorporation was quantified by measuring the optical density (OD) at 450 nm.
    Trypan blue exclusion assay was performed to determine cell viability after treatment with nor-wogonin, wogonin, or wogono-side. TNBC cells (MDA-MB-231, BT-549, HCC70, and HCC1806) and non-tumorigenic breast cells (MCF-10A and AG11132) cells/well) were plated (5 103/well) in 96-well plates and treated with nor-wogonin, wogonin, wogonoside, or DMSO for 24 h. The cultured  cells were harvested and resuspended in 100 ml of RPMI 1640 medium, DMEM, or MEGM and the cell suspension was thoroughly mixed with an equal volume of 0.4% trypan blue solution (Gibco, Grand Island, NY, USA). The numbers of viable and dead cells were counted using a hemocytometer, and the cell viability percentage was determined.
    Cell cycle analyses
    Cell cycle arrest of MDA-MB-231 cells was analyzed by a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) using the BD PharmingenTM FITC-BrdU flow kit (BD Biosciences), according to the manufacturer’s instructions. Briefly, MDA-MB-231 cells were treated with different concentrations of nor-wogonin (10, 20, or 40 mM), or DMSO for 24 h. For the time course experiments, MDA-MB-231 cells were treated with nor-wogonin (30 mM) or DMSO for 24, 48, and 72 h. After treatment, cells were labeled by incubating overnight with BrdU solution at a final concentration of 10 mM in cell culture medium. After labeling, cells were fixed, permeabilized using BD Cytofix/Cytoperm Buffer and incubated with BD Cytoperm Permeabilization Buffer Plus for 10 min on ice and washed by 1X BD Perm/Wash Buffer. Then, cells were fixed again using BD Cytofix/Cytoperm Buffer and incubated with DNase for 1 h at 37 C to expose incorporated BrdU. Cells were then washed with BD Perm/Wash Buffer and resuspended in 50 ml of BD Perm/Wash Buffer containing diluted fluorescein isothiocyanate (FITC)-labeled anti-BrdU antibody and incubated for 20 min at room temperature. Cells were then washed with 1 ml of BD Perm/Wash Buffer and resuspended in 20 ml of the 7-aminoactinomycin D (7-AAD) solution. Finally, cells were resuspended in 1 ml of staining buffer and analyzed using a FACS Calibur flow cytometer. Quantitative analysis of the FACS data was carried out using the FlowJo software (FlowJo, Ashland, OR, USA)
    Apoptosis analyses
    Induction of apoptosis of MDA-MB-231 cells by nor-wogonin was investigated by the FACS Calibur flow cytometer (BD Biosciences) using the annexin V/propidium iodide (PI) staining kit (BioLegend, San Diego, CA, USA), according to the manufac-turer’s instructions. MDA-MB-231 cells were treated with nor-wogonin, nor-wogonin combined with 30 mM pan-caspase inhibitor (Z-VAD-FMK), or DMSO for 24 h. Cells were then harvested, washed twice with PBS, resuspended in annexin V binding buffer, and stained with annexin V-FITC and PI at room temperature in the dark for 30 min. The MDA-MB-231 cells were then analyzed by flow cytometry using quadrant statistics for apoptotic and necrotic cell populations. The fluorescence intensity in X-axis and Y-axis were detected in the FL1-A and FL2-A channels respectively. Annexin V was used to detect both the early and late stages of apoptotic cells while PI was used to detect late apoptotic and necrotic cells. Quantitative analysis of the FACS data was performed using the FlowJo software.