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  • br NP mM Tris HCl pH containing


    1% NP-40, 50 mM Tris-HCl [pH 7.5]) containing 0.1 mM Na2VO3, 1 mM NaF, and protease inhibitors (Sigma). For immunoblotting, proteins from whole cell lysates were resolved by 10% or 12% SDS-poly-acrylamide gel electrophoresis (PAGE) and then transferred to ni-trocellulose membranes. Primary DETA NONOate were used at 1:1000 or 1:2000 dilutions. Secondary antibodies conjugated with horseradish peroxidase were used at 1:2000 dilutions in 5% nonfat dry milk. After final washing, nitrocellulose membranes were exposed for an enhanced chemiluminescence assay using the LAS 4000 mini (Fuji, Tokyo, Japan).
    5.7. Sphere formation assay
    Cells were cultured in 24-well ultra-low attachment plates in serum-free medium supplemented with 5 g/mL insulin, 0.4% bovine serum albumin, 10 ng/mL basic fibroblast growth factor, and 20 ng/mL human recombinant epidermal growth factor for 6 d. The size and number of spheroids were analyzed under an optical microscope. Spheroids more than 50 mm in size are the criteria for sphere forma-tion.
    5.8. Wound-healing assay
    Cells (1 × 105 cells/well) were plated in a growth medium in 24-well plates and incubated for 24 h. After the formation of a complete monolayer was confirmed, the cells were wounded by scratching lines using a standard 200-μL plastic tip. Migration and cell movement throughout the wound area were observed by a phase-contrast micro-scope after 24 h.
    5.9. Transwell invasion assay
    Cell invasive capacity of A549RT-eto (5 × 104 cells per well) was determined using a Transwell Filter (8 μm pore size, Corning) with matrigel (BD Biosciences). Briefly, PC cells treated with a CX combi-nation were transferred in each upper chamber in 200 μL of serum-free medium, before 500 μL of complete medium was added into each bottom chamber with the same concentration of curcumin. After in-cubation for 24 h, the cells in the upper chamber were removed, and the invaded cells in the membrane were stained with hematoxylin–eosin. The stained cells in at least six randomly selected fields were photo-graphed and counted under an optical microscope.
    5.10. Short interference RNA transfection
    Cells were trypsinized and incubated overnight to achieve 60%–70% confluency before siRNA transfection. Human NF-κB siRNAs (commercially pre-made at Bioneer, Daejeon, Korea; 100 nM) or nega-tive control siRNAs (Bioneer; 100 nM) were mixed DETA NONOate with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cells were incubated with the transfection mixture for 24 h and then rinsed by the RPMI-1640 medium containing 10% fetal bovine serum. The cells were incubated
    C. Kaewpiboon et al.
    for 48 h before harvest.
    5.11. Luciferase reporter assay
    A549RT-eto cells were transfected with Rel A-luciferase or pGL3 empty vector as a control luciferase vector. To normalize the transfec-tion efficiency, a pGK-βgal vector that expresses galactosidase from a phosphoglucokinase promoter was included in the transfection mixture. CX was added to the transfected cells at 12 h before harvest. At 48 h post-transfection, cells were washed with cold PBS and lysed in a lysis solution (25 mM Tris [pH 7.8], 2 mM EDTA, 2 mM DTT, 10% glycerol, and 1% Triton-X100). The luciferase activity was measured with a lu-minometer using a luciferase kit (Promega, Madison, WI, USA).
    5.12. Statistical analysis
    Data are presented as means ± standard deviation (S.D.). The Student’s t-test was used for statistical analysis, with p value < 0.05 being significant.
    This study was supported by IPST for research funds for DPST graduate with first replacement (No.03/2557) and the Korea Institute for Advancement of Technology (KIAT) in Republic of Korea (N0002310, Construction Project of Supporting Center for Commercializing Customized Nano-mold-based Technologies).
    Appendix A. Supplementary data
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