Anti αv

HEC-1A, Ishikawa and RL95-2 cells were grown on 96-well plates, and treated with rhPAPPA, anti-PAPPA (Santa Cruz, USA), different human serum samples or different conditional media of JAR cells. After cells formed a confluent monolayer, mouse IgG, anti-αV, anti-β3, anti-αVβ3 (Santa Cruz, USA); Three Lectins: Ulex europaeus agglutinin (UEA-1), Lotus tetragonolobus lectin (LTL) and Lensculinaris agglutinin (LCA) (Vector Laboratories, USA) were added to block the specific epitope for 4 h. JAR cells were stained with CellTracker™ Green CMFDA (Invitrogen, USA) for 1 h before the adhesion assay. The stained cells (1 × 10⁴) were plated onto treated HEC-1A, Ishikawa or RL95-2 cell monolayers in JAR cell culture medium. After 1 h, unattached JAR cells were removed, and the attached cells were gently washed with PBS 3 times. The cells were than photographed under a fluorescent microscope (Olympus, Japan). An equal amount of stained JAR cells (1 × 10⁴) was plated in 3 blank wells. After detection using a multimode plate reader (PerkinElmer, USA), adhesion rate was calculated as a percentage of attached JAR cells. All experiments were replicated 3 times.

A375 cells were plated in DMEM culture media at a density of 1 × 10⁴ cells per well in an 8-chambered microscope slide. Cells were allowed to adhere for 4 hours before removal of media and replacement with media containing vehicle (PB) or 100 μM Lunasin. Cells were allowed to incubate with treatment media for up to 24 h. At selected times, cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. Cells were incubated at -20°C in 100% methanol before blocking with 1% bovine serum albumin. Cells were incubated with anti-Lunasin (1:1000) rabbit polyclonal antibody and anti-α_V (1:100) mouse monoclonal antibody (Santa Cruz #376156) in blocking solution. Following overnight incubation, cells were washed and incubated with appropriate secondary antibodies conjugated to AlexaFluor-488 or AlexaFluor-647 fluorophores (Jackson ImmunoResearch). After washing, mounting media containing DAPI (Thermo Fisher) was dropped onto slides, and the slides were sealed using a 60 mm cover slip and clear finger nail polish prior to fluorescent analysis. Images were taken on a Nikon NiE upright microscope using Nikon Elements software (Nikon).