Supernatant was then removed and the pellet was washed with ice-cold

PBS and centrifuged again at 4°C for 5 minutes at 2000 rpm. This pellet was then resuspended in ice-cold RIPA buffer (Upstate Cell Signaling Solutions, Temecula, CA) containing Complete Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and centrifuged at 14,000 rpm for 15 minutes at 4°C. Supernatant containing total cell protein was collected and stored at -80°C. 3H-Thymidine Cell Proliferation Assay Cell proliferation was measured by3H-thymidine incorporation into T24 human bladder cancer cells, plating 1.5 ×103 cells/well onto a 96-well cell culture plate (Corning Incorporated), www.selleckchem.com/products/Trichostatin-A.html in 150 μL/well McCoy’s 5A medium containing 10% heat inactivated FBS, 1% antibiotic/antimycotic solution, 1% L-glutamine, and plus 2.2 grams/L sodium bicarbonate. The next day, cell growth MEK162 medium was removed and replaced with 100 μl serum-free McCoy’s medium. On the third day, synthetic as -APF was resuspended

in acetonitrile/distilled water (1:1) and applied to the cells in serum-free McCoy’s medium at varying concentrations; cell controls received acetonitrile/distilled water diluted in serum-free McCoy’s medium (same final concentration of diluent). Cells were then incubated at 37°C in a 5% CO2 atmosphere for an additional 48 hours, after which they were labeled with 1 μCi per well3H-thymidine at 37°C in a 5% CO2 atmosphere for 4 hours. The cells were then treated with trypsin-EDTA (Invitrogen), insoluble cell contents harvested and methanol-fixed onto glass fiber filter paper, and the amount of radioactivity incorporated determined using a Beckman scintillation counter. Significant inhibition of3H-thymidine incorporation was defined as a decrease in cpm of >2 SD from the mean of Proteasome inhibitor control cells for each plate.

Real-time qRT-PCR Gene expression was determined using SYBR® Green based real-time RT-PCR, QuantiTect® primers and reagents (Qiagen) and a Roche 480 LightCycler. Samples were tested in triplicate runs, and specific mRNA levels quantified and compared to mRNA levels for β-actin or GAPDH using Roche LC480 real-time PCR analysis software (version 1.5.0). Predetermined optimal concentrations of RNA were Montelukast Sodium used for each set of primers. p53 (QT00060235), Akt (QT00085379), GSK3β (QT00057134), β-catenin (QT00077882), MMP2 (QT00088396), GAPDH (QT01192646), and β-actin (QT1680476) primer sets were obtained from Qiagen. p53 served as a standard control for APF activity, while GAPDH and β-actin served as standard controls for the qRT-PCR procedure. SDS Polyacrylamide Gel Electrophoresis and Western Blot Assay Specific protein expression or phosphorylation was determined by Western blot. Protein concentration was measured using a Folin reagent-based protein assay kit (Bio-Rad, Hercules, CA).