Event EBV-related transformation and the proliferation of human B cells.blood mononuclear cells (PBMCs) derived from the whole blood of 7 healthy volunteers and cord blood mononuclear cells (CBMNCs) VX-509 obtained from 3 anonymous donors were isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). Primary B cells were purified from the PBMC fractions of 3 donors by negative selection using magnetic beads Miltenyi Biotec (Gladbach, Germany) according to the manufacturer’s specifications. These primary cells were cultured in RPMI 160 medium supplemented with 20 FBS and 1 penicillin and streptomycin designated herein after as culture medium. In experiments using PBMC, the culture medium was supplemented with Cyclosporine A (500 nM/ml; Sigma, Marlborough, MA) to inhibit T-cells immunity.EBV-transformation AssaysThree different approaches were used to investigate the effects of resveratrol on B cell EBV transformation efficiency. First, PBMCs, CB-MNC (26106 cells/mL) or purified B cells (0.56106 cells/mL) were exposed to a defined virus dose (50 moi) for two hours at 37uC and then seeded in replicate wells of 96-well plates in medium containing several concentrations of resveratrol or vehicle (DMSO 0.05 ). In a second set of experiments, cells were infected with a range of virus dilutions before being seeded into a 96-well plate in the presence or absence of resveratrol (50 mM). In another assay, cells were exposed to a defined virus dose (50 moi) and then seeded at three-fold limiting dilutions into replicate wells of a 96-well plate. Fresh medium containing the drug was replaced once per week. In all cases, the percentage of wells containing EBV-transformed cell clones was assessed with microscopic inspection six weeks after infection. Infection experiments for protein and mRNA analysis were performed by exposing purified B cells (16106) to EBV and then harvesting the cells at different time points post-infection. LCLs were generated from PBMCs (2 donors) and one CB-MNC donor by infecting 26106 cells with the supernatant of B95-8EBV and expanded into 25 ml flasks. Cultures were maintained in exponential growth using passaging twice weekly. LCLs were treated with sodium butyrate (5 mM) for 3 days in some experiments, to induce lytic infection and their RNA was extracted and used for quantitative real time PCR.Materials and Defactinib web Methods Cell LinesThe EBV producing cell line B95-8 was obtained from the American Type Culture Collection (Rockville, MD) and they were cultured in RPMI 160 medium supplemented with 10 FBS and 1 penicillin and streptomycin. The Akata cell line carrying the EGFP-EBV [16], which is a Burkitt lymphoma-derived cell line [17] were obtained from Dr K. Takada (Hokkaido University). These cells were cultured in RPMI 160 medium supplemented with 10 FBS and 500 mg/ml G418 as described [16].ReagentsResveratrol and anti-a tubulin antibody were purchased from Sigma. Hygromycin was obtained from Wako (Tokyo Japan). The NFkB luciferase report vector pGL4.32 [-luc2PNFkB E/ Hygro] was acquired from Promega. The antibodies directed against survivin, Mcl-1, phosphorylated STAT-3, histone H3 and phosphorylated p65 were purchased from Cell Signaling Technology. Anti-EBNA1 clone 1B5 (Acris Herford, Germany), antiEBNA2 clone PE2, anti-human IgG and anti-LMP1 1655472 clone CS1-4 were from DakoCytomation (Glostrup, Denmark). Recombinant proteins including IL-4, IL-10, soluble CD40 ligand and TNFa were purchased from Pep.Event EBV-related transformation and the proliferation of human B cells.blood mononuclear cells (PBMCs) derived from the whole blood of 7 healthy volunteers and cord blood mononuclear cells (CBMNCs) obtained from 3 anonymous donors were isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, Oslo, Norway). Primary B cells were purified from the PBMC fractions of 3 donors by negative selection using magnetic beads Miltenyi Biotec (Gladbach, Germany) according to the manufacturer’s specifications. These primary cells were cultured in RPMI 160 medium supplemented with 20 FBS and 1 penicillin and streptomycin designated herein after as culture medium. In experiments using PBMC, the culture medium was supplemented with Cyclosporine A (500 nM/ml; Sigma, Marlborough, MA) to inhibit T-cells immunity.EBV-transformation AssaysThree different approaches were used to investigate the effects of resveratrol on B cell EBV transformation efficiency. First, PBMCs, CB-MNC (26106 cells/mL) or purified B cells (0.56106 cells/mL) were exposed to a defined virus dose (50 moi) for two hours at 37uC and then seeded in replicate wells of 96-well plates in medium containing several concentrations of resveratrol or vehicle (DMSO 0.05 ). In a second set of experiments, cells were infected with a range of virus dilutions before being seeded into a 96-well plate in the presence or absence of resveratrol (50 mM). In another assay, cells were exposed to a defined virus dose (50 moi) and then seeded at three-fold limiting dilutions into replicate wells of a 96-well plate. Fresh medium containing the drug was replaced once per week. In all cases, the percentage of wells containing EBV-transformed cell clones was assessed with microscopic inspection six weeks after infection. Infection experiments for protein and mRNA analysis were performed by exposing purified B cells (16106) to EBV and then harvesting the cells at different time points post-infection. LCLs were generated from PBMCs (2 donors) and one CB-MNC donor by infecting 26106 cells with the supernatant of B95-8EBV and expanded into 25 ml flasks. Cultures were maintained in exponential growth using passaging twice weekly. LCLs were treated with sodium butyrate (5 mM) for 3 days in some experiments, to induce lytic infection and their RNA was extracted and used for quantitative real time PCR.Materials and Methods Cell LinesThe EBV producing cell line B95-8 was obtained from the American Type Culture Collection (Rockville, MD) and they were cultured in RPMI 160 medium supplemented with 10 FBS and 1 penicillin and streptomycin. The Akata cell line carrying the EGFP-EBV [16], which is a Burkitt lymphoma-derived cell line [17] were obtained from Dr K. Takada (Hokkaido University). These cells were cultured in RPMI 160 medium supplemented with 10 FBS and 500 mg/ml G418 as described [16].ReagentsResveratrol and anti-a tubulin antibody were purchased from Sigma. Hygromycin was obtained from Wako (Tokyo Japan). The NFkB luciferase report vector pGL4.32 [-luc2PNFkB E/ Hygro] was acquired from Promega. The antibodies directed against survivin, Mcl-1, phosphorylated STAT-3, histone H3 and phosphorylated p65 were purchased from Cell Signaling Technology. Anti-EBNA1 clone 1B5 (Acris Herford, Germany), antiEBNA2 clone PE2, anti-human IgG and anti-LMP1 1655472 clone CS1-4 were from DakoCytomation (Glostrup, Denmark). Recombinant proteins including IL-4, IL-10, soluble CD40 ligand and TNFa were purchased from Pep.
