induced apoptosis, including the caspase-12/caspase-4, CHOP/ GADD153, IRE1/PERK/JNK, and p53 signaling pathways [19,20,21,22]. p53 tumor suppressor is a nuclear protein that functions as a regulator of transcription and mediates several biological effects, such as growth arrest, senescence, and apoptosis in response to various forms of stress [23]. Elevation of p53 expression during ER stress in MEFs, MCF-7 and HCT116 cell lines has been reported [22]. p53 has been demonstrated to play an important role in the dysregulation of ER [24,25]. Although elevation of p53 gene expression during ER stress has been described, it has been unclear whether and how p53 gene expression is regulated in response to ER stress. In addition, UPR has been shown to regulate glycogen synthase kinase (GSK) 3b which is essential for the regulation of p53 and cyclin D1 degradation during early ER stress [26,27], and p53 protein was downregulated at 3–6 h with tunicamycin or brefeldin A treatment. Because ER dysfunction has been linked to many diseases, it is important to investigate the mechanism by which p53 expression is regulated during ER stress. It is interesting to note that NF-kB plays a role in p53 expression in certain situations [28]. NF-kB may specifically recognize an NF-kB site on the p53 promoter and activates the p53 promoter [29]. Because ER stress activates NF-kB, we hypothesized that ER stress induces p53 expression may through NF-kB activation. In this report, we demonstrate ER stress induced by tunicamycin and brefeldin A leads to increased expression of p53 and that the increased expression of p53 is mediated by NF-kB. Furthermore, when we knocked down p53 expression by p53 RNAi, the cells were more resistant to tunicamycin and Brefeldin A treatment. Therefore, in these ER stress conditions, induction of p53 is required to induce cell death. Taken together, expression of p53 in response to ER stress is regulated by NF-kB, and participates in ER stress-induced cell death. The NF-kB appears to play dual roles in regulating cell survival and death under ER stress. Materials and Methods Chemicals Tunicamycin, Brefeldin A, ethidium bromide, diethyl pyrocar- bonate, actinomycin D and SDS were obtained from Sigma. Bay 11-7082 and cycloheximide were purchased from Calbiochem. ECL Western blot detection system was from Amersham Biosciences. The RT-PCR reagent, proTaq plus, oligo(dT)15 primer, M-MLV-Reverse transcriptase, dNTP, and G418 were purchased from Promega (Madison, WI). The NE-PER nuclear and cytoplasmic extraction reagents kit and Micro BCATM protein assay reagent kit were from Pierce. Anti-GRP78 was purchased from Transduction Laboratories. The anti-p53 was purchased from Oncogene (Merck Ltd., Budapest, Hungary). The following antibodies from Cell Signaling (Beverly, MA) were used: anti-p-Ser15-p53, anti-p-Ser20-p53, anti-p-Ser37-p53, and anti-p- Ser46-p53 antibodies. Anti-IkBa, anti-Bax, anti-MDM2, anti-p65, and anti-NF-kB p50 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-c-tubulin was from MDBio (Frederick, MD). Topoisomerase II was from NEOMAKERS. Anti-b-actin was from Chemicon (Pittsburgh, PA). LipofectA- MINE 2000, Dulbecco’s modified Eagle’s medium (DMEM), and antibiotic mixture (10,000 units of penicillin, 10,000 mg of streptomycin) and trypsin-EDTA were products of Invitrogen. Fetal bovine serum was obtained from Biological Industries (Beit Haemek, Israel). Regulation of p53 Expression by NF-kB in ER Stress Cell Culture MCF-7 and Hela cell lines were obtained from ATCC (Manasses, VA, USA). MCF-7 pSuper stable transfectant, and MCF-7 pSuper p53 stable transfectant transfectant cell lines were generated form MCF-7 and HCT116, and these cell lines were maintained at 37uC in a 5% CO2 atmosphere in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin. Plasmid and Stable Clone Cell Lines Construction pSuper and pSuper p53 siRNA plasmid were provided gratis from Brummelkamp. The original pSUPER vector is the is the H1 RNA polymerase III promoter, which drives the endogenous production of shRNA. is the H1 RNA polymerase III promoter, which drives the endogenous production of shRNA. is the H1 RNA polymerase III promoter, which drives the endogenous production of shRNA. H1 RNA polymerase-III promoter to direct intracellular synthesis of siRNA like transcripts. The two pLKO.1- shRNA vectors used for knockdown of NF-kB p65 are the following: TRCN0000014684 (shRelA) and TRCN000014686 (shRelA). The two shRelA vectors were obtained from National RNAi Core Facility (Taipei, Taiwan). MCF-7 cells were co- transfected with pCDNA3.1/ pSuper or pcDNA3.1/pSuper p53 plasmids by using Invitrogen LipofectAMINE 2000 reagent according to the manufacturer’s protocol. Cells were then selected by G418 for 2 weeks. The pCDNA3.1/ pSuper and pcDNA3.1/ pSuper p53 stable clone cell lines were established by western blotting. Preparation of Cytosolic and Nuclear Extracts Subcellular fractionation was performed using NE-PER Nucle- ar and Cytoplasmic Extraction Reagents kit according to the manufacturer’s instructions. In brief, MCF-7 (16106) cells in 10- cm dishes were exposed to 1 mg/ml Brefeldin A in 10% FBS– containing DMEM for 0, 6, 12, and 24 h. After treatment, the cells were washed with cold calcium and magnesium free PBS, collected with a cell scraper, and harvested by centrifugation. The cell pellets were suspended in 200 mL of Cytoplasmic Extraction Reagent I solution and incubated on ice for 10 min, followed by adding 11 mL of Cytoplasmic Extraction Reagent II solution and incubation on ice for 1 min. The cell suspensions were centrifuged at 16,0006g for 5 min to collect the supernatant cytoplasmic fraction. The pellets were resuspended with 100 mL of Nuclear Extraction Reagent on ice for 40 min. This suspension was centrifuged at 16,0006g for 10 min at 4uC to collect the supernatant nuclear fraction. Western immunoblotting Cells, seeded in 10-cm dishes (16106 cells per dish), were incubated for 16 h, subjected to different drug treatments, and harvested by scraping. Cell lysates were prepared by treating cells with 2X SDS lysis buffer (0.1 M Tris (pH 6.8), 0.4% SDS, and 20% glycerol). The protein concentration of the supernatant was measured using a Micro BCATM protein assay reagent kit. To the cell lysate, the same volume of SDS-PAGE sample loading buffer [100 mmol/L Tris-HCl, 4% SDS, 5% b-mercaptoethanol, 20% glycerol, and 0.1% bromophenol blue (pH 6.8)] was added, and the cells were boiled for 10 min. Equal amounts of proteins were resolved in SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Pierce). Following blocking with 5% nonfat dry milk for 1 h at room temperature and washing with Tween 20 with Tris-buffered saline (TTBS), the polyvinyli- PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e39120