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  • br Materials and methods br Reagents and antibodies br BMS

    2020-08-02


    Materials and methods
    Reagents and antibodies
    BMS-777607 was purchased from MedChemExpress (Mon-mouth Junction, NJ). Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DAPI, propidium iodide (PI), Liu'sstain solution A and B were purchased from Sigma. Antibodies against c-Met, p-c-Met (Y1234/Y1235), Gab1, p-Gab-1, Gab2, p-Gab2, PI3K, p-PI3K, AKT, p-Akt, p-c-raf, cyclinA2, cyclinB1, cyclinD1, p-histone H3, Aurora A, Aurora B, a-tubulin, and b-actin were purchased from Cell Signaling Technologies (Beverly, MA, USA). An Annexin V apoptosis detection kit with PI was purchased from Biolegend (San Diego, CA).
    Mouse lines and cell lines
    NU/NU female mice were purchased from BioLASCO, Taiwan. The animals were housed under specific pathogen-free conditions. All procedures were conducted in accordance with approved pro-tocols and recommendations for the appropriate care and use of 
    laboratory animals. Human ovarian cancer cell lines, namely SKOV3, ES-2, TOV112D, TOV21G, A2780, OVCAR3, and OVCAR8, were obtained from the American Type Culture Collection. The cell lines were cultured in RPMI medium supplemented with 10% (v/v) fetal bovine serum, 100 units/mL of penicillin, and 100 units/mL of streptomycin at 37 C in a humidified atmosphere containing 5% CO2.
    Western blot analysis
    Western blot analysis for specific protein detection was per-formed according to a previously mentioned protocol. Briefly, 50 mg of cell total lysate protein was resolved using a 7.5% SDS-PAGE gel and electrotransfered to a polyvinylidene difluoride membrane. The membrane was blocked in blocking buffer with 5% skim milk, after which it was probed with the primary LOXO-101 diluted ac-cording to the manufacturer's instructions and incubated overnight at 4 C. The membrane was further incubated using relevant sec-ondary antibodies with conjugated horseradish peroxidase. Sub-sequently, the membrane was incubated with an enhanced chemoluminescence substrate, and the signal was detected using a chemoluminator.
    Cell viability analysis
    The cells were seeded in a 96-well plate at a density of 5 103 cells/well and exposed to various concentrations of BMS-7776007 for 72 h. At the end of the treatment, the cells were incubated in MTT reagent for 3 h and the precipitated dye was solubilized in 100% DMSO. The absorbance of each well was determined using a microplate reader at 570 nm, with the reference wave length being 690 nm. The viability of the cells was calculated as the ratio of the absorbance from treated samples to that of the untreated control sample.
    Apoptosis assay
    The cells were treated with 2 10 5 M BMS-7776007 for 24, 48, and 72 h and stained using the Annexin V apoptosis detection kit with PI. The proportion of apoptotic cells was determined using flow cytometry.
    Cell migration assay
    Cell migration was determined using an in vitro “wound-heal-ing” assay. Briefly, the cells were seeded in ibidi chambers (culture inserts) (2 104 cells/window) and cultured overnight. The culture inserts were removed the next day, and the cells were treated with 2 10 5 M BMS-7776007. Wound closure was monitored using a light microscope. Images were recorded at 0 and 24 h by using a light microscope fitted with a charge-coupled device camera.
    Cell cycle analysis
    The cells were treated with 2 10 5 M BMS-7776007 for 48hand fixed in chilled ethanol overnight. After washing with 1 phosphate-buffered saline (PBS), the cells were further incu-bated in 1 PBS with 20 mg/mL of PI and 20 mg of RNase A for 30 min at room temperature (RT). The DNA content was analyzed using a FACSC aliburflow cytometer (BD Biosciences, DNA Staining Protocol for Flow Cytometry).
    Liu's staining
    Ten thousand SKOV3 cells were seeded on a cover slip and cultured overnight for attachment. The cells were then treated with 2 10 5 M BMS-777606 for 48 h. The cells were rinsed with PBS and stained with Liu's stain according to the manufacturer's in-structions. The slides were then rapidly but gently rinsed under running tap water. They were then dried and examined under a light microscope.
    Immunofluorescence staining
    Twenty thousand SKOV3 cells were seeded on a cover slide and cultured overnight for attachment. The cells were then treated with 2 10 5 M BMS-777606 for 48 h and then fixed in cold methanol ( 20 C).The cells were treated for 30 min at RT with an anti-a-tubulin antibody to stain a-tubulinin of the cells. After rinsing with PBS, the slips were further incubated with PE-conjugated second-ary antibodies for 30 min at RT. The cells were counter stained with DAPI, and the cover slips were mounted using antifade mounting media (Sigma). The fluorescent images were visualized and recor-ded using a Zeiss Axioplan2 imaging microscope (Carl Zeiss,