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  • br Materials and methods br Cell culture and transfection

    2019-10-21


    2. Materials and methods
    2.1. Cell culture and transfection
    Human breast cancer cell (MDA-MB-231 and SK-BR-3) was pur-chased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). MDA-MB-231 cells were maintained in RMPI-1640 medium while SK-BR-3 cells were maintained in DMEM medium which were both supplemented with 10% heat activated FBS and Methoxy-X04 (100 U/mL penicillin/streptomycin) at 37 °C in 5% CO2. Cells were seeded into 6-well plates at a density of 90% and incubated overnight. Lipofectamine™ 6000 (Invitrogen, Carlsbad, CA, USA) was used for cell transfection according to the manufacturer's instructions.
    MTT assays were applied to assess the viability of cells treated with different groups of compounds. The groups of compounds were as fol-lows: SM934 alone; Testosterone alone; the combination of SM934 and Testosterone and SM934-Testosterone. The results were calculated as percentages of the absorbance (λ = 492, 630 nm) of the control group. The half inhibition concentration (IC50) indicates the concentration of compound at which the survival rate of cells Methoxy-X04 reaches 50%. MTT was purchased from Sigma Chemical (St Louis, MO, USA).  European Journal of Pharmacology 858 (2019) 172382
    2.3. Colony formation assay
    Breast cancer cells MDA-MB-231 and SK-BR-3 were seeded in 24-well plates at 500 cells/well with different drugs. Seven days later, 4% Paraformaldehyde Fix Solution was used to fixate the cells, and stain the colonies by crystal violet.
    2.4. Wound-healing assay
    Cells were seeded in 6-well plates. After cells were converged around 90%, then sterile 10 μl pipette tips were used to scratch the cells. Washed the cells with PBS and added the serum-free medium with different groups of compounds. The wound was photographed with a microscope at h, 24 h, and 48 h, respectively.
    2.5. Migration and invasion assays
    Transwell assays were performed to verify the metastasis ability of breast cancer cells. Cells were treated with different compounds and were cultured in 200 μl serum-free medium in the upper chamber with (invasion) or without (migration) Matrigel. The DMEM medium with 10% heat activated FBS was added in the under chamber with the same drugs. The cells were incubated for 48 h at 37 °C with 5% CO2, and then the membrane of the chamber was stained with 4% Paraformaldehyde Fix Solution and photographed using a fluorescence microscope (Nikon, Japan).
    2.6. Western blot analysis
    Total proteins of breast cancer cells were extracted by using RIPA Lysis Buffer, and Bradford assay (Pierce Biotechnology Inc., Rockford, USA) was used to evaluate the total protein concentrations. Western blot assay was performed as described previously (Chen et al., 2017). The following antibodies were used: CTSK (DF6614, Affinity Bios-ciences, OH, USA); β-actin (60008-1-Ig, Protein Tech Group, China). β-Actin was used as the internal loading control.
    2.7. Quantitative real-time PCR analysis
    The total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) and reverse-transcribed into cDNA by Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) assays were conducted using Hieff™ qPCR SYBR® Green Master Mix (No Rox) and a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA, USA). CTSK mRNA was normalized to GAPDH mRNA. The primer se-quences were as follows: CTSK, forward: 5′-TTCTGCTGCTACCTGTG GTG -3′ and reverse: 5′-CCAGGTGGTTCATAGCCAGT -3′; GAPDH, for-ward: 5′-CAGGGCTGCTTTTAACTC-3′ and reverse: 5′-GGAAGATGGTG ATGGGAT-3′ (Liang et al., 2018). The results were calculated using the 2-△△CT method, and the results are expressed as the mean ± S.D.