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  • br Materials and methods br


    2. Materials and methods
    Human CRC cell lines HCT116 and SW480 were from the Type Culture Collection, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium with 10% FBS (both from Gibco-BRL, Gaithersburg, MD, USA) in a humidified incubator at 37 °C containing 5% CO2.
    C.-e. Wu, et al. Experimental Cell Research xxx (xxxx) xxxx
    Fig. 3. Cinnamaldehyde and oxaliplatin suppress CRC K 252a under normoxia. Both types of CRC were incubated with CA and oxaliplatin separately or sy-nergistically under normoxia condition. (A) Cell viability of different cells were tested by MTT assay. (B) mRNA expression of apoptosis (Bax, Bcl-2), Wnt signaling (β-catenin, Cyclin-D1), EMT (E-cadherin, N-cadherin), stemness (CD133, CD44) related markers were studied by RT-PCR. (C) Protein expression of apoptosis (cleaved Caspase-3, cleaved PARP1), Wnt signaling (β-catenin, Cyclin-D1), EMT (E-cadherin, N-cadherin), stemness (CD133, CD44) related molecules were researched by Western Blot. *P < 0.05, **P < 0.01.
    Cinnamaldehyde was from China National Institute for the Control of Pharmaceutical and Biological Products (purity 99%). Annexin V/PI kit was from BD Biosciences (San Diego, CA). TRIzol reagent and Power SYBR Green PCR Master Mix were from Life Technologies (Grand Island, NY, USA). PrimeScript RT reagent kit with gDNA Eraser was from TaKaRa (Dalian, China).
    CRC cell lines were seeded into 96-well plates at the density of 7 × 103/per well, followed by incubation with different drugs. 20 μL MTT solution (5 mg/ml) was used at 37 °C for 4 h and 150 μL DMSO was added into per well. The absorbance was studied at an OD of 490 nm by a microplate reader (Bio-Tek, Winooski, VT, USA). Cell inhibitive rates were calculated using the formula: 1-ODexperiment/ODcontrol. The ex-periment was repeated for three times.
    CRC cells were inoculated onto cover slips in a 6-well plate at a density of 2 × 105 cells per well and incubated for 24 h before being treated with different drugs. Cells were then fixed with 4% paraf-ormaldehyde for half an hour and then washed once with PBS. After that, cells were stained with 50 ng⁄mL Hoechst 33258 for half an hour. Apoptotic morphological modifications were characterized as con-densed or fragmented nuclei. Nuclear modifications were detected by an Axioplan2 fluorescence microscope (Zeiss, Jena, Germany).
    2.5. Apoptosis assay
    Cell apoptotic rate was examined by Annexin V/PI method. Cells were seeded in 6-well plates to be treated with drugs and then collected 
    after trypsinization. Cells were resuspended in 500 μL binding buffer and stained with 5 μL annexin V-FITC and PI for 15min in dark. The analysis was then performed by FCM.
    2.6. Spheroid formation assay
    CRC cells (1 × 103/well) were seeded into ultralow attachment six-well plates filled with serum free medium contains 2% B27, 20 ng/ml EGF and bFGF at 37 °C in 5% CO2. Medium was changed every 6 days. Clony formation pictures were photographed 20 days after incubation.
    Cells were trypsinized and tumor tissues were homogenized. Total RNA was extracted from CRC cells or tissues using TRIzol reagent and then reverse transcribed into cDNA by TaKaRa RT reagent kit. Gene expression was studied by ABI 7500 fast RT-qPCR System (ABI; Applied Biosystems, Waltham, MA, USA) via DNA-binding dye SYBR-Green through △△Ct method. ACTB was selected to be internal reference.
    2.8. Western blot assay
    CRC cells and tissues were lysed by RIPA buffer containing protease inhibitor cocktail (P8340, Sigma-Aldrich). The lysate was then cen-trifuged at 12000g for 20 min at 4 °C. The concentration of protein was measured using Bradford method and the samples were stored at −80 °C until experiment. Equal amounts of protein (20 μg) from each group were separated by SDS-PAGE, followed by transferring to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were incubated with BSA for 1 h and probed with primary antibodies at 4 °C overnight and then with secondary antibodies for 1 h. Species reactivity of all the primary antibodies used was only to human. Protein levels were de-tected by ECL kit. The information of the primary antibodies used is listed below: Bax (CST, #5023), Bcl-2 (CST, #4223), Cleaved Caspase-3