Genomic DNA was isolated from cells using Trizol, and 500 ng grnomic DNA was bisulfite modified using the EZ DNA Methylation Gold Kit according to the manufacturers protocols. Two proce dures were used. First, methylation status was analyzed by Bioactive compound bisulfite modified DNA sequencing of the corre sponding CpG islands. Six independent clones were ana lyzed. The PCR was performed using a Rotor Gene 3000 with 45 cycles of denaturation for 30 s and annealing for 60 s, and a final extension at 72 C for 4 min. PCR products were subcloned into T easy vector for sequencing. Western blot analysis Cells were washed with ice cold PBS and lysed in ice cold RIPA on ice for 30 min. Total protein was measured using Bio Rad protein assay reagent according to the manufacturers protocol.

Protein was seperated by 10% PAGE gels and transfered to Polyvinylidene Fluoride membranes. After wash ing with tris buffered saline, the membranes were blocked with 5% bovine serum albumin /phosphate buffered saline The membranes were washed three times with PBS and then incubated with peroxidase linked secondary antibody for 1 h at room temperature. The signals were developed using an ECL kit, scanned, and analyzed with Total Lab software. The relative expression of target proteins was presented as the ratio to B actin. Cell invasion assay Cell invasion was assessed by using a BD BioCoat Matrigel Invasion Chamber according to the manufacturers instructions. Cells were loaded into chamber inserts containing an 8 um pore size membrane with a thin layer matrigel matrix.

Cells migrating to the lower surface of the membrane during 48 h were fixed with 100% methanol. The membranes were then stained with hematoxylin, scanned, and ana lyzed with an Aperio Scanscope System. Flow cytometry of cell cycle Cells were fixed with 70% ethanol for 72 h and stained with 25 ug/mL propidium iodide in fluorescence activated cell sorting buffer for 30 min at room temperature in the dark, the cells were analyzed by flow cytometry using a Becton Dickinson FACScan. Experiments were performed in triplicate in three independent experiments. Proliferation assay Cells were cultured in phenolred free medium containing 5% charcoal stripped FBS. Cell proliferation was analyzed every 24 h via colorimetric assay with 3 2, 5 diphenyltetrazolium bromide. Absorbance at 490 nm was evaluated by a Spectra Max 190 microplate reader.

Experiments GSK-3 were performed in triplicate in three independent experiments. Soft agar colony assay Cells were seeded in 0. 3% top agar in growth medium over a layer of 0. 6% agar in a 6 well plate at a density of 1 104 cells/well. After 3 weeks of incubation, colonies with more than 50 cells were counted and photographed with an inverted microscope. The assay was performed at least three times in triplicate.