Sub-G1 cells in flow cytometric histograms were considered apoptotic cells. Analysis of cell cycle distribution and the percentage

of cells in the G1, S, and G2/M phases of the cell cycle were determined using the software FlowJo (Tree Star, Ashland, OR). To detect apoptosis, the Annexin V–FLUOS kit (Roche Diagnostics) was used. Cells were treated for 6, 24, or 48 hours with saffron extract. After washing twice in phosphate-buffered saline, 1 × 106 cells were stained with 100 μL annexin V staining solution, consisting of 20 μL fluorescein isothiocyanate–conjugated RXDX-106 manufacturer annexin V reagent (20 μg/mL), 20 μL isotonic propidium iodide (PI; 50 μg/mL), and 1000 μL of 1 mol/L HEPES buffer, for 15 minutes at room temperature. Cells were analyzed on a FACSCalibur flow cytometer (Becton-Dickinson) using a 488 nm excitation and 530/30 nm band-pass filter for fluorescein detection and a long-pass filter 2P670 nm for PI detection after electronic compensation. Because positive annexin V staining indicates apoptotic STI571 nmr and necrotic cells, PI-positive cells were used to measure late apoptotic cells and necrotic cells, whereas annexin V–positive and

PI-negative cells were counted as early apoptotic cells. Whole-cell lysates were prepared from HepG2 tumor cells. Protein concentration of lysates was determined with a Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA), and 30 μg proteins were loaded onto 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. The gels were transferred to nitrocellulose membranes before immunodetection processing with anti-phospho-H2AX (Millipore), anti-caspase-3 (Cell Signaling Technology), anti-IκB (Abcam, Cambridge, UK), anti-TNFR1 (Santa Cruz Biotechnology, Santa Cruz, CA), and with secondary antibodies (anti-mouse or anti-rabbit IgG peroxidase conjugated; Pierce, Rockford, IL). Bound antibodies were detected by incubating the blots in West Pico chemiluminescent substrate (Pierce). The level of

however immunoreactivity was measured as peak intensity using an image-capture and analysis system (GeneGnome; Syngene, UK). Hybridization with anti-GAPDH was used to control equal loading and protein quality. SPSS (version 10) statistical program (SPSS Inc., Chicago, IL) was used to carry out a one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analysis of differences between the means of the treated and control groups were performed by using Dunnett’s t test. Other experimental procedures are described in detail in the supporting information. These include animal housekeeping and treatment, in vitro antioxidant properties, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-ending labeling (TUNEL) assay, immunohistochemical analyses, morphology and histopathology analysis, as well as enzyme-linked immunosorbent assay (ELISA).