dene fluoride membranes were incubated overnight at 4uC with primary antibody in TTBS containing 1% bovine serum albumin. The second anti-mouse antibody-horseradish peroxidase conju- gate (1:2000 dilutions) was subsequently incubated with mem- branes for 1 h at room temperature and washed extensively for 40–50 min with TTBS at room temperature. The blots were probed with the ECL Western blot detection system according to the manufacturer’s instructions. RT-PCR and real-time PCR analysis of p53 mRNA After treatment, the cells were washed with cold PBS and then cells were harvested. Total RNA was extracted from MCF-7 cells using TRIzol reagent (Invitrogen) and chloroform extraction. RT was performed using 2 mg of total RNA. cDNA synthesis was performed using 200 U of Moloney murine leukemia virus reverse transcriptase, 5 mM oligoDT, 1 mM dNTP solution, and 3 mM Mg2+ in a volume of 20 ml. The PCR reaction was performed using the following primers: p53 gene: (F) 59-GAGGTTGGCTCTGACTGTACC-39/(R) 59-CCTCATTCAGCTCTCGGAA C-39. GRP78 gene: (F) 59- TAGCGTATGGTGCTGCTGTC-39/(R) 59-GTCAGGC- GATTCTG GTCATT-39. GAPDH gene: (F) 59-CCATCAAT- GACCCCTTCAT-39/(R) 59-TGGACTGTGT CATGAGTCC- 39. The products were visualized after electrophoresis on a 1.5% agarose gel containing ethidium bromide. The signal level of the bands was quantified densitometrically. In real-time PCR, PCR was performed for the resulting RT products using oligonucleotide primers specific for p53, and GAPDH. The primers used were as follows: p53 gene (F) 59-GCG CACAGAGGAAGAGAATC-39/ (R) 59-CCTCATTCAGCTCTCTCGGAAC-39; GAPDH gene (F) 59-GATTCCACCCATGGCAAATTC-39/(R) 59-AG- CATCGCCCCACTT GATT-39. All PCR reactions were performed with ABI 7500 Fast Real-Time PCR System using DNA-binding SYBR Green dye for detection of the PCR products and results were analyzed by ABI StepOne Software version 2 (Applied Biosystems). Chromatin immunoprecipitation assay (CHIP) MCF-7 cells were plated into 100 mm-diameter dishes for overnight incubation and stimulated with or without 1 mg/ml of Brefeldin A for 0, 3, 9, and 24 h. The CHIP assay was performed according to the supplier’s directions (Millipore-Upstate, Billerica, MA). Briefly, treated cells were fixed in serum-free medium containing 1% formaldehyde for 10 minutes at 37uC. MCF-7 cells were cross-linked with 1% formaldehyde for 10 min. The cells were then harvested, lysate, and sonicated to shear genomic DNA into fragments of 200–1000 base pairs. An aliquot was also retained as an input sample to normalize PCR reactions and analyze shearing efficiency. 400 mg of each remaining chromatin preparation was incubated overnight at 4uC with 2 mg of anti-NF- kB p50, anti-NF-kB p65, and anti-p53 antibodies. Both immunoprecipitated and input DNA samples were analyzed by PCR to determine the relative amounts of DNA from the p53 gene promoter region present in the samples. The primers used for amplification by PCR were: (F) 59CAC CAG GTC GGC GAG AAT CCT39/(R) 59CTC TAG ACT TTT GAG AAG CT39. The PCR products were visualized after electrophoresis on a 1.5% agarose gel containing ethidium bromide. Electrophoretic mobility shift assay (EMSA) EMSA were conducted using LightShift chemiluminescent EMSA kits (Pierce). Briefly, nuclear extracts (5 mg total protein) were incubated with approximately 15 pmol of biotinylated oligonucleotides probe corresponding to the NF-kB consensus Regulation of p53 Expression by NF-kB in ER Stress sequences in p53 promoter (59- TGGGATTGGGGTTTTCCCCTCC-39, containing the kB site from the p53 promoter) in 25 mmol l–1 Hepes (pH 7.6), 100 mmol l–1 NaCl, 15 % glycerol, 0.1 % NP-40 and 0.5 mmol l–1 PMSF in a final volume of 20 ml. Incubations were conducted for 60 min at room temperature. After incubation, mixtures were applied to 5 % acrylamide non-denaturing gels and electrophoresed for approx- imately 2 h at 100 V. Gels were then transferred to nylon membranes via electroblotting at 380 mA for 30 min. NF-kB–p53 probe complexes on each blot were visualized by a chemilumines- cent reaction with streptavidin/horseradish peroxidase, according to the manufacturer’s protocol, and visualized with the BioSpec- trum AC imaging system (UVP, CA) according to the manufac- turer’s instructions. Immunofluorescence MCF-7 cells were seeded at 26105 per well in 6-well flat- bottomed plates and incubated in 10% FBS–supplemented DMEM for 24 h. Cells were treated with 2.5 mg/ml tunicamycin or 1 mg/ml Brefeldin A in the same medium. Cells for immunofluorescence microscopy of NF-kB and p53 were fixed with 3.7% paraformaldehyde for 10 min and washed three times with PBS. Cells were then treated with ice-cold methanol for 2 min and washed three times with PBS. Cells were stained for NF-kB and p53 translocation using anti-p65 and p53 antibodies overnight at 4uC and then anti-mouse FITC-conjugated antibody for 1 h. After staining with antibody, cells were viewed with a fluorescence microscope. Cell viability Assay Cell viability was assessed with a 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium- bromide (MTT) assay in three replicates. MCF-7 and stable transfectant cells were seeded at 36105 per well in 24-well flat-bottomed plates and incubated in 10% FBS– supplemented DMEM for 24 h. Cells were treated with 5 mg/ml Tunicamycin and 1 mg/ml BFA in the same medium. Controls received DMSO vehicle at a concentration equal to that in drug- treated cells. After 48 h, the drug-containing medium was replaced with 200 mL of 10% FBS–supplemented DMEM containing 0.5 mg/mL MTT, and cells were incubated in the CO2 incubator at 37uC for 4 h. Medium was removed, the reduced MTT was solubilized in 600 mL per well of DMSO, and 100 mL aliquots from each well was transferred to 96-well plates to measure absorbance at 570 nm. Luciferase reporter assay The p53 and NF-kB transcriptional activity was performed by luciferase reporter assay. MCF-7 cells were transfected with pp53-TA-Luc (p53 response element) or NF-kB-responsed pro- moter plasmid (NF-kB response element) by using Invitrogen LipofectAMINE 2000. After 16 h incubation, the cells were treated with or without Tunicamycin or Brefeldin A at the time indicated. The luciferase activity was measured using a Promega luciferase assay system according to the manufacturer’s protocol. The firefly luciferase activity was normalized to that of Renilla luciferase. Statistical analysis Results were presented as the mean 6 S.D., and statistical comparisons were made using the Student’s t test. Significance was defined at the p,0.05 or 0.01 levels. PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e39120