Were plated in 96-well plates and cultured for 72 hours in the presence or absence of resveratrol and Indolactam V assessed for viability and proliferation using an MTT colorimetric assay (Roche, Indianapolis, IN, USA). The inhibition rate of cell proliferation was calculated by the formula: inhibition = (absorbance of control cells ?absorbance of treated cells)/absorbance of control cells6100.Establishment of EBV-positive Cells Lines Stably Expressing an NFkB Luciferase VectorThe pGL4.32 [-luc2PNFkB E/Hygro] vector, designated as NFkB-Luc, which contains five copies of an NFkB response element and drives transcription of the luciferase reporter gene was used to examine NFkB activity in EBV-infected cells lines. The NFkB-Luc vector was transfected into two LCL cells using the GenomONETM-Neo EX HVJ Envelope Transfection Kit (Ishihara Sangyo, Tokyo Japan) according to the manufacturer’s protocol. Stable transfectants were established by selection with hygromycin B (300 mg/ml).LDH AssayPurified B cells (26105 cells/ml) from three donors were suspended in RPMI 1640 phenol free medium supplemented with 2.5 BSA and 1 FBS, plated in 96-well plates and cultured for 12, 24, 48 and 72 hrs in the presence or absence of several concentrations of resveratrol. The release of LDH in the cells supernatants was measured using the LDH cytotoxicity detection kit (Roche).NFkB -Luciferase Activation AssayThe cell lines stably expressing the NFkB-Luc vector were cultured for 6 hrs in the presence or absence of variable concentrations of resveratrol. Some cells were pretreated with 20 ng/ml of TNF-a to over activate NFkB signal. The cells were harvested and lysed using the passive lysis buffer 18297096 (Promega). The luciferase activity in the lysates which represents NFkB activity was quantified using a luciferase system (Promega) in a Mabskan LB system (Thermo Scientific).Resveratrol Prevents EBV-Transformation of B CellsFigure 2. Resveratrol does not block the entry of EBV into B cells but induces apoptosis in EBV infected cells. (A) Primary B cells were infected with EBV-GFP and cultured in the presence or absence of resveratrol (50 mM). The infected cells were collected several times after infection and the percentage of cells expressing EGFP (EBV-infected B cells) was assessed using flow cytometry. Summarized data from three different donors is shown. (B) Purified B cells were infected with EBV-GFP and cultured for up to 96 hours with vehicle or the indicated dose of resveratrol and examined using flow cytometry to MedChemExpress 301353-96-8 quantify the proportion of apoptotic cells. Summarized data using B cells from three different donors is shown and the error bars are the means6SEM of the percentage of apoptotic cells (C) EBV-GFP-infected B cells were treated with 50 mM of resveratrol and cultured for the indicated times and the number of cells in apoptosis was determined using flow cytometry. A representative result of three independent experiments is shown. (D) B cells were infected with EBV and cultured for 1379592 72 hours with or without resveratrol and the levels of cytokines released into the culture medium were measured using ELISA or an ELISA multianalyte assay. Mean6SEM of three independent experiments is shown. doi:10.1371/journal.pone.0051306.gWestern Blot AnalysisWhole-cell protein samples were prepared by lysing the cells in M-PERH Mammalian Protein Extraction Reagent (Pierce) supplemented with phosphatase and a protease inhibitor cocktail (Sigma). Nuclear and cytoplasmic fractions we.Were plated in 96-well plates and cultured for 72 hours in the presence or absence of resveratrol and assessed for viability and proliferation using an MTT colorimetric assay (Roche, Indianapolis, IN, USA). The inhibition rate of cell proliferation was calculated by the formula: inhibition = (absorbance of control cells ?absorbance of treated cells)/absorbance of control cells6100.Establishment of EBV-positive Cells Lines Stably Expressing an NFkB Luciferase VectorThe pGL4.32 [-luc2PNFkB E/Hygro] vector, designated as NFkB-Luc, which contains five copies of an NFkB response element and drives transcription of the luciferase reporter gene was used to examine NFkB activity in EBV-infected cells lines. The NFkB-Luc vector was transfected into two LCL cells using the GenomONETM-Neo EX HVJ Envelope Transfection Kit (Ishihara Sangyo, Tokyo Japan) according to the manufacturer’s protocol. Stable transfectants were established by selection with hygromycin B (300 mg/ml).LDH AssayPurified B cells (26105 cells/ml) from three donors were suspended in RPMI 1640 phenol free medium supplemented with 2.5 BSA and 1 FBS, plated in 96-well plates and cultured for 12, 24, 48 and 72 hrs in the presence or absence of several concentrations of resveratrol. The release of LDH in the cells supernatants was measured using the LDH cytotoxicity detection kit (Roche).NFkB -Luciferase Activation AssayThe cell lines stably expressing the NFkB-Luc vector were cultured for 6 hrs in the presence or absence of variable concentrations of resveratrol. Some cells were pretreated with 20 ng/ml of TNF-a to over activate NFkB signal. The cells were harvested and lysed using the passive lysis buffer 18297096 (Promega). The luciferase activity in the lysates which represents NFkB activity was quantified using a luciferase system (Promega) in a Mabskan LB system (Thermo Scientific).Resveratrol Prevents EBV-Transformation of B CellsFigure 2. Resveratrol does not block the entry of EBV into B cells but induces apoptosis in EBV infected cells. (A) Primary B cells were infected with EBV-GFP and cultured in the presence or absence of resveratrol (50 mM). The infected cells were collected several times after infection and the percentage of cells expressing EGFP (EBV-infected B cells) was assessed using flow cytometry. Summarized data from three different donors is shown. (B) Purified B cells were infected with EBV-GFP and cultured for up to 96 hours with vehicle or the indicated dose of resveratrol and examined using flow cytometry to quantify the proportion of apoptotic cells. Summarized data using B cells from three different donors is shown and the error bars are the means6SEM of the percentage of apoptotic cells (C) EBV-GFP-infected B cells were treated with 50 mM of resveratrol and cultured for the indicated times and the number of cells in apoptosis was determined using flow cytometry. A representative result of three independent experiments is shown. (D) B cells were infected with EBV and cultured for 1379592 72 hours with or without resveratrol and the levels of cytokines released into the culture medium were measured using ELISA or an ELISA multianalyte assay. Mean6SEM of three independent experiments is shown. doi:10.1371/journal.pone.0051306.gWestern Blot AnalysisWhole-cell protein samples were prepared by lysing the cells in M-PERH Mammalian Protein Extraction Reagent (Pierce) supplemented with phosphatase and a protease inhibitor cocktail (Sigma). Nuclear and cytoplasmic fractions we.
