Regulation of p53 Expression by NF-kB in ER Stress Figure 4. Induction of NF-kB activity during ER stress. (A) The effect of BFA on IkBa expression in MCF-7 cells. The cells were incubated with 1 mg/ml Brefeldin A in 10% FBS–supplemented DMEM for 0, 3, 6, 12, and 24 h. The total cell lysates were analyzed by Western blotting with antibodies for IkBa and b-actin. (B) The effect of 1 mg/ml brefeldin A on NF-kB phosphorylation in MCF-7 cells. The cells were incubated with 1 mg/ml BFA as time indicated. The whole cell lysates were subjected to immunoblotting with antibodies against anti-p65, anti-p-ser276-p65, anti-p-ser311-p65 and b-actin. (C) Analysis of NF-kB activity during ER stress in MCF-7 cells. The cells were transfected with reporter vectors encoding firefly luciferase driven by NF-kB-induced promoter, and transfectants were treated with BFA as time indicated. Luciferase activities were normalized to that of cotransfected Renilla luciferase. Columns, mean of three independent experiments; bars, SD (*, P,0.05, Student’s t test). (D) Nuclear NF-kB DNA binding activity was measured by EMSA using a probe corresponding to the NF-kB-binding site of the p53 promoter. The arrows indicated the NF-kB complexes, and the supershifted band was induced with anti-p65 subunit or anti-NF-kB p50 subunit antibody.(E) ER stress induced NF-kB subunits p50, p65, and p53 binding to p53 gene promoter in MCF-7 cells. MCF-7 cells were treated with 1 mg/ml BFA for for 0, 3, 9, and 24 h. Nuclear lysates were generated and immunoprecipitated with anti-NF-kB p50, anti-NF-kB p65, and anti-p53 antibodies. Immunoprecipitated DNA was anayzed for p53 promoter by PCR and gel electrophoresis. 4% of input served as loading control. doi:10.1371/journal.pone.0039120.g004 as indicated. Application of LiCl in our experiment was shown to successfully block GSK-3b activity by increasing phosphorylation of GSK-3b at ser9. However, even when GSK-3b is inactivated, induction of p53 expression was still observed (Fig. 6). Therefore, this result showed that GSK-3b does not involve in regulation of p53 expression in response to the prolonged ER stress. Induction of p53 is required for ER stress-induced cell death To address how important the induction of p53 is to the ER stress-induced cell death, we transfected pSuper-siRNA-p53 vector into MCF-7 cells to knockdown p53 expression. Two lines of stable transfectants were generated and named as pSuper-p53 RNAi-1 and pSuper-p53 RNAi-2. The efficiency of p53 gene silencing was evaluated using western blotting. Transfectants pSuper-p53 RNAi-1 appeared to have a better knockdown effect than pSuper-p53 RNAi-2 (Fig. 7A, upper panel). Also, these transfectants, which expressed pSuper-siRNA-p53 construct, were shown to efficiently reduce both basal and ER stress-induced level of p53 protein (Fig. 7A, lower panel). To study the effect of p53 knockdown in ER stress, we compared the morphology changes between MCF-7 cells and their transfectants following tunicamy- cin treatment. In the MCF-7 and MCF-7 pSuper vector control cells, tunicamycin treatment resulted in a dose-dependent, pro- gressive change in cell shape from flat to round, while in the transfectants pSuper-p53 RNAi-1 and pSuper-p53 RNAi-2, tunicamycin is more limited to cause this change (Fig. 7B). Finally, an MTT assay was applied to measure cell survival after ER stress was induced. Treatment of MCF-7 cells and MCF-7 pSuper vector control cells with tunicamycin or brefeldin A for 48 hours induced severe cell death. In contrast, knockdown of p53 prevented cells from tunicamycin or brefeldin A-triggered cell death (Fig. 7C), thereby indicating that p53 is indispensible in the process of ER stress-induced cell death. Discussion Prolonged ER stress leads to cell apoptosis. Several signal transduction pathways have been identified that can explain how PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e39120