The samples were analyzed by using a flow cytometer (EasyCyte MINI – Guava Technologies). Immunoblots The medium was removed after the treatments, and the cells were washed with PBSA and lysed with RIPA buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 2 mM EGTA]. The lysates were centrifuged and the supernatants were collected. 30 μg of protein were

fractionated by SDS-PAGE on a 10% gel, and transferred to a PVDF membrane (Amersham Bioscience). A blocking solution (5% BSA (containing the phosphatase inhibitors NaF and orthovanadate)) was added to the membrane for 1 hour. The membrane was incubated overnight with an anti-p53 or anti-phospho-p53 Adriamycin chemical structure (Ser15) (Abcam Inc.) antibodies diluted at 1:300. The immune complexes were detected by using the ECL Western blotting

detection kit (Amersham Pharmacia). The ImageJ program was used for the densitometric analyses. M30, tubulin and actin staining Cells were plated on coverslips (3 × 105 cells/35 × 11 mm dishes). After 48 h of treatment, the cells were fixed with formaldehyde 3.7% for 30 minutes, washed with PBSA and treated with ribonuclease (10 mg/mL). To detect cytokeratin 18 fragments we added M30 antibody (FITC-conjugated) (CytoDEATH-Roche Labs) overnight at room temperature. The cells were submitted to immunofluorescence with anti-α and β-tubulin (Sigma, 1:200) overnight at room temperature and secondary antibody anti-mouse TRITC-conjugated. In some cases, actin cytoskeleton was analyzed by using phalloidin this website FITC-conjugated and anti-α Pexidartinib datasheet and β-tubulin with secondary antibody anti-mouse CY5-conjugated (Invitrogen, 1:200). Nuclei were counterstained with propidium iodide (10 μg/mL). The images were analyzed by Laser Scanning Confocal Microscopy (Zeiss- LSM510) and we counted 1,000 cells/slide. Nuclear abnormalities frequency Cells

were plated on coverslips (3 × 105 cells/35 × 11 mm dishes), grown for 24 h and treated with cinnamic acid at different concentrations. After 48 h of treatment, the cells were fixed with formaldehyde 3.7% for 30 minutes, treated with ribonuclease (10 mg/mL) for 30 minutes and stained with propidium iodide (10 μg/mL) during 20 minutes. We analyzed 2,000 cells/coverslips and the nuclear aberrations (micronucleation, binucleation and multinucleation) were counted according to the classification of Tolbert et al. [39], modified by Manelli-Oliveira and Machado-Santelli [40]. Statistics Statistical analysis on cell viability was achieved by χ2 tests to determine a statistical difference between the treated cells and the control group for each concentration. Flow cytometry, BrdU incorporation, protein expression, M30 labeling and nuclear aberrations data were analyzed by using the two way ANOVA test to verify a possible concentration-response or time-response relationship. We also analyzed cell death by using Multidimensional Nonlinear Descriptive Analysis (estimation by using negative binomial model).