Clin. studies not only discovered a unique anticancer drug candidate but also shed new light on the understanding of ROS generation and function and the potential application of a ROS-promoting strategy in cancer treatment. antibodies were from BD Biosciences. Mouse anti-phospho-STAT3, mouse anti-caspase 3 antibodies, and rabbit anti-poly(ADP-ribose) polymerase antibodies were from Rabbit polyclonal to ZNF138 Cell Signaling Technology, Inc. Determination of Cellular ROS Accumulation of intracellular ROS was detected with the probe DCFH2-DA as described previously (16). In brief, after drug treatment, cells were labeled with 10 m DCFH2-DA (2,7-dichlorofluorescin diacetate) for 20 min at 37 C in a humidified atmosphere at 5% CO2. The labeled cells were washed and collected. To quantify ROS, the fluorescence intensity (FL-1 channel) was measured by flow cytometry (FACSCalibur, BD Biosciences). Cell Viability Assay About 5000 cells/well were seeded into 96-well plates. Twenty-four hours later, cells were treated with vehicle control or various concentrations of NPP, PEITC, menadione, or taxol for 72 h. After various treatments, 20 l of MTT solution (5 mg/ml, Sigma Aldrich) was added to each well and incubated at 37 C for 3 h. The supernatant was aspirated, and the MTT-formazan crystals were dissolved in 150 l of dimethyl sulfoxide. The absorbance was measured by a microplate reader (Molecular Devices) at a wavelength of 570 nm. Immunoblotting Analysis Whole cell lysates were prepared in 1 Laemmli sample buffer (Sigma) to eCF506 extract total proteins. Equivalent amounts of total cellular protein were electrophoresed on an 8% SDS-PAGE gel and transferred onto nitrocellulose membranes (Millipore). Membranes were blocked in 5% nonfat milk in TBS containing 0.1% Tween 20 (TBST) for 1 h at room temperature and then incubated with primary antibodies in 5% BSA in TBST at 4 C eCF506 overnight. Membranes were then washed with TBST and incubated with HRP-conjugated secondary antibody in 5% BSA in TBST for 1 h at room temperature. Immune complexes were detected by enhanced chemiluminescence (Pierce). RNAi and Transfection TP53 siRNA-1 5-GACUCCAGUGGUAAUCUACdTdT-3, TP53 siRNA-2 5-CUACUUCCUGAAAACAACGdTdT-3, and a random sequence control siRNA were purchased from Genepharma (Shanghai, China). Synthetic siRNAs were transfected into LO2 cells using Lipofectamine 2000 (Invitrogen). Real-time Quantitative PCR Assay The mRNA abundances of antioxidant genes were determined by quantitative real-time PCR assays. The ??Ct method of relative quantification and SYBR Green chemistry were used, and -actin was used as an endogenous control for normalization. PCR primer sets were designed using Primer Premier 5, and the sequences were as follows: TP53, 5-AGAATCTCCGCAAGAAAGG-3 (forward) and 5-CAAGCAAGGGTTCAAAGAC-3 (reverse); CDKN1A, 5-ACTTTGATTAGCAGCGGAACA-3 (forward) and 5-CTGTCCATAGCCTCTACTGC-3 (reverse); SESN2, 5-AAGACCACCCGAAGAATGT-3 (forward) and 5-AGGAGTCAGGTCATGTAGCG-3 (reverse); SOD1, 5-GCTGGTTTGCGTCGTAG-3 (forward) and 5-CCTTCGTCGCCATAACT-3 (reverse); SOD2, 5-GGACAAACCTCAGCCCTAA-3 (forward) and 5-TGAAACCAAGCCAACCC-3 (reverse); GPX1, 5-GTCGGTGTATGCCTTCTCGG-3 (forward) and 5-CAGCTCGTTCATCTGGGTGT-3 (reverse); GPX4, 5-AGAACGGCTGCGTGGTG-3 (forward) and 5-TTGTGGAGCTAGAAATAGTGGG-3 (reverse); Bcl-2, 5-TCCAATCCTGTGCTGCTA-3 (forward) and 5-ACTCTGTGAATCCCGTTT-3 (reverse); Bcl-xL, 5-CGTGGAAAGCGTAGACAA-3 (forward) and 5-GTGGGAGGGTAGAGTGGAT-3 (reverse); and -actin, 5-TCCCTGGAGAAGAGCTACG-3 (forward) and 5-GTAGTTTCGTGGATGCCACA-3 (reverse). Luciferase Assay HepG2/STAT3 cells (1.5 105 cells/well) were seeded into eCF506 24-well cell culture microplates (Corning), allowed to grow for 24 h, and then treated with reagents for 2 h followed by stimulation with 10 ng/ml IL-6 for 5 h. Equal numbers of cells were collected, and the luciferase activity was measured by a luminometer using a luciferase assay system (Promega). All luciferase assay experiments were performed at least three times to minimize the differences caused by cell numbers. Assessment of Apoptosis NPP-induced apoptosis was determined by an annexin V-FITC apoptosis detection kit (KeyGen). Briefly, MDA-MB-468 cells were harvested after exposure to NPP for 24 h. The cells were washed twice with cold PBS and then resuspended in 500 l of binding buffer at a concentration of 1 1 106/ml. Cells were then stained with annexin eCF506 V-FITC and PI and analyzed with a FACScan flow cytometer (BD Biosciences). Viable cells were negative for both PI and annexin V. Apoptotic cells were positive for annexin V and negative for PI, whereas late apoptotic cells and necrotic cells displayed both high annexin V and PI labeling..