After blocking incubation in 0.5% bovine serum albumin (BSA) in 1 × phosphate-buffered Emricasan saline (PBS) for 10 min, we labeled the cells with PE-conjugated anti-human E-Cadherin antibody (BioLegend, San Diego, CA) for 1 h, followed by DNA staining using 7-AAD Viability Dye (Beckman Coulter, Indianapolis, IN) for 5 min. Control cells were labeled with PE-conjugated mouse IgG1, κ isotype

ctrl (BioLegend). We then analyzed the E-cadherin expression on the cells using the Epics XL-MCL™ Flow Cytometer (Beckman Coulter). Data are presented as the median, mean, and mode of fluorescence intensity of the cells counted. Western blotting The cells treated under the same conditions as those for flowcytometry were lysed in lysis buffer (50 mM Tris pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton-X100) containing 1 mM PMSF, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mU/ml aprotinin, 50 mM sodium fluoride, 2 mM sodium orthovanadate, and 50 nM Calculin A (Cell signaling). The protein concentration in the cell lysates was check details determined by the Bradford protein assay (Bio-Rad). Twenty μg of total proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham). The membranes were blocked with 5% skim milk in PBS containing 0.1% Tween 20, and probed with mouse anti-E-cadherin

antibody (BD Biosciences) at 1:1000 dilution overnight at 4°C. Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG sheep antibody (Amersham) for 1 h. The reactive proteins were visualized using ECL-plus (Amersham) according to the manufacturer’s instructions. Equal loading of proteins was confirmed by probing the membranes with mouse anti- β-actin antibody (Sigma). Immunofluorescent staining HSC-2 cells for immunofluorescent staining of E-cadherin were seeded in slide chambers (IWAKI, Tokyo, Japan) and treated with 25 μM of celecoxib

or DMSO for 24 h. After washing the cells extensively with PBS, we fixed the cells with cold methanol for 10 min at -20°C. After washing with PBS, the cells were incubated with Alexa Fluor 488-conjugated anti-E-cadherin antibody (Santa Cruz Biotechnology, Dallas, TX) at 1:200 dilution in PBS for 1 h. The nuclei were visualized by staining with Hoechst 33258 Evodiamine (Sigma-Aldrich). Stained cells were then mounted with Prolong Gold Antifade Reagent (Invitrogen). The fluorescent images were captured through a fluorescence microscope (Olympus, Tokyo, Japan). Patients and tissue samples Human tissue specimens were obtained from patients with histologically verified tongue squamous cell carcinoma (TSCC) who underwent primary surgery at the Department of Otorhinolaryngology–Head and Neck Surgery, Keio University Hospital (Tokyo, Japan) between 2003 and 2011. Informed LY2835219 clinical trial consent from patients and approval from our Institutional Ethics Review Board were obtained for the use of the clinical materials in the present study.