The experimental protocols were approved by the Ethics Committee of the Institute of Biomedical Sciences, University of São Paulo, Brazil (Protocol CEP-ICB n. 308/09). Cinnamic acid Cinnamic acid (CAS

number 140-10-3) was obtained as trans-cinnamic acid crystals, 99 + % (Sigma Aldrich Chemical Company Inc.) and the solutions were prepared by using 24 mg of the compound and 500 μL of ethanol. Phosphate buffered saline (PBSA) was added to complete 10 mL (final PD0332991 datasheet concentration at 16 mM). An appropriate control with DMEM, 20% PBSA and 1% ethanol was used. Cytotoxicity assay The MTT kit (Promega) was used to evaluate the cytotoxicity. Briefly, 1 × 104 cells were seeded in each well containing 100 μL of DMEM plus 10% of FBS in a 96-well plate. After 24 h, various concentrations of cinnamic acid were added. The control group received drug-free medium. After 2 days, 15 μL of “Dye Solution” were added to each well and the Selleck Tariquidar plates were incubated for additional www.selleckchem.com/products/Liproxstatin-1.html 4 h. Then, 100 μL of “Solubilization/Stop Solution” were added in each well and the optical density was measured at 570 nm in an ELISA plate reader (BIO-RAD). Propidium iodide staining for flow cytometry NGM and HT-144 cells (3 × 105 cells/35 × 11 mm dishes) were incubated for 24 h and

then treated with different concentrations of cinnamic acid. After 2 days the cells were harvested and submitted to fixation with 75% of ice-cold methanol at 4°C for 1 h. Cells were then washed with PBSA and suspended in propidium iodide staining solution containing 200 μL of PBSA, 20 μL of ribonuclease (10 mg/mL) and 20 μL of propidium iodide (10 μg/mL). The cell suspensions were incubated for 1 h at 4°C and 5,000 cells were analyzed by flow cytometry in each group (EasyCyte MINI – Guava Technologies). 5-bromo-2-deoxyuridine incorporation After incubation and treatment with cinnamic acid the cells were submitted to BrdU (50

μM) (Sigma) incorporation for 30 minutes or 1 hour at 37°C. The samples Molecular motor were washed with PBSA and fixed with ethanol/acetic acid (3:1) for 15 minutes. The cells were incubated with HCl 2 M for 30 minutes. Then, we added antibody anti-BrdU (Sigma) (1:100) for 1 hour and, then, secondary antibody FITC-conjugated for 30 minutes. The cells were treated with ribonuclease (10 mg/mL) and the nuclei were counterstained with propidium iodide (10 μg/mL). We analyzed 1,000 cells/coverslips. Activated-caspase 9 assay NGM and HT-144 cells (3 × 105 cells/35 × 11 mm dishes) were incubated for 24 h and subsequently treated with different concentrations of cinnamic acid. After 6, 12 or 24 hours the cells were harvested and suspended at 1 × 105 cells/mL. Then, we added Caspase Reagent Working Solution (protocol by Guava Technologies) into the cell suspension. After incubation for 1 hour at 37°C we added 100 μL of 1× Apoptosis Wash Buffer in each sample and centrifuged them at 300 G for 7 minutes.