Immunoblotting Briefly, 70–80% confluent cells were homogenized with 1 ml of lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF) and incubated on ice. To the homogenates was added 125 μl of 10% NP-40 solution, and the mixture was then centrifuged for 30 sec at 12,000 × g. Supernatant protein concentration was determined by the Bradford GSK461364 manufacturer protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (Sigma) as a standard. Immunoblot analysis was performed as described elsewhere . Immunofluorescence analysis and confocal microscopy Cells grown on coverslips were fixed in 4% PFA, permeabilized
in 0.3% Triton X-100, and blocked for 40 min in 1% BSA/10% fetal bovine serum. The cell samples were incubated with CHIR98014 clinical trial primary antibodies at 4°C overnight, washed with PBS containing 0.1% BSA, and then reacted with FITC- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA,
USA) at room temperature for 40 min. After washing, the samples were rinsed with PBS containing 0.1% selleck screening library BSA, stained with 5 mg/ml 4,6-diamidino-2-phenylindole (DAPI; Sigma), and mounted. Confocal analyses were performed using an Olympus (Center Valley, PA) FC-300 Confocal Laser Scanning Microscope equipped with FITC- and Cy3- channel filter systems. All images were converted to TIFF format and arranged using Photoshop 7.0 (Adobe, Seattle, WA). In vitro migration assay The in vitro migration assay was performed as described previously . 5 × 104 cells were placed in the upper compartment (8 μm pore size) of the cell culture insert with Fenbendazole or without 5 μM PIA. Medium, supplemented with 100 ng/ml IGF-I (R&D Systems, Minneapolis, MN), was added to the lower compartment. After 12 h of incubation, the cells on the upper surface of the filter were wiped out with a cotton swab, and the filter was removed from the chamber and stained with Diff-Quick stain set (Fisher, Pittsburgh, PA). The migration of the cells was determined by counting the number of cells that migrated through the pores to the lower side of the
filter under a microscope at 100 × magnification. We performed the assay three times, and three randomly selected fields were counted for each assay. We used Student’s t test to determine the significance at a level of P < 0.05. Results Screening of oral squamous cell carcinoma cell lines We screened several OSCC cell lines in order to select suitable cell line models with the characteristics of the EMT (low or negative expression of E-cadherin) and a constitutively activated state of Akt. Of the 7 OSCC cell lines, KB, KOSCC-25B, Ca9-22, and SCC-15 showed constitutively activated phosphorylated Akt (p-Akt). Of these four lines, only KB and KOSCC-25B showed low or negative expression of E-cadherin (Fig. 1A). Because the E-cadherin downregulation could be caused by the methylation of its promoter, we investigated the methylation status of E-cadherin gene promoter in the KB and KOSCC-25B cells with MS-PCR.