Apoptotic and necrotic cells were stained using an apoptosis detection kit (Invitrogen), as recommended by the manufacturer, and analyzed using a flow cytometer (FACSCanto; BD Bioscience, San Jose, CA, USA). apoptosis. The induction of apoptosis required the ODNs to contain the CpG motif and the expression of TLR9 in lymphoma B cells. A decrease in Bcl-xl expression and an increase in Fas and Fas ligand expression accompanied lymphoma B-cell apoptosis. Treatment with the Fas ligand-neutralizing antibody inhibited CpG ODN-induced apoptosis. CpG ODN brought on a transient NF-B activation in the Timosaponin b-II B-cell lymphoma cell collection, which constitutively expresses a high level of c-Myc, while CpG ODN induced sustained increases in NF-B activation and c-Myc expression in main B cells. Furthermore, an NF-B inhibitor inhibited the proliferation of the CH27 B-cell lymphoma collection. Our data suggest that the differential responses of lymphoma and main B cells to CpG ODN are the result of differences in NF-B activation. The impaired NF-B activation in the CpG ODN-treated B-cell lymphoma cell collection alters the balance between NF-B and c-Myc, which induces Fas/Fas ligand-dependent apoptosis. mRNA using reverse transcription polymerase chain reaction (RT-PCR). Mononuclear cells were isolated from your spleens of BALB/c and C57BL/6 mice (Charles River Laboratories, Inc., Frederick, MD, USA) by Ficoll (Sigma-Aldrich, St Louis, MO, USA) density gradient centrifugation. B cells were isolated by T-cell depletion using anti-Thy 1.2 antibody (BD Bioscience, San Diego, CA, USA) and guinea pig match (Rockland Immunochemicals Inc., Gilbertsville, PA, USA). Timosaponin b-II The producing cells were panned to remove monocytes and dendritic cells. All experiments involving animals have been examined and proved by the Institution Animal Care and Use Committee at University or college of Maryland (R-07-41 and R-10-87). RT-PCR analyses of tlr9 mRNA TRIzol reagent (Invitrogen, MCM2 Carlsbad, CA, USA) was used to purify RNA from two CH27 clones and splenic B and T cells as recommended by the manufacturer. cDNA was generated from this RNA using SuperScript II reverse transcriptase (Invitrogen). Tlr9 mRNA levels were assessed by PCR amplification with specific primers and Taq DNA polymerase (Invitrogen) and the following cycling conditions: 94 C for 30?s, 55 C for 30?s and 68 C for 1?min for 25 cycles. The -tubulin gene was amplified as a control using the following cycling conditions: 94 C for 30?s, 56 C for 30?s and 68 C for 1?min for 25 cycles. The primers specific for were 5-GCACAGGAGCGGTGAAGGT-3 Timosaponin b-II and 5-GCAGGGGTGCTCAGTGGAG-3, and the -tubulin-specific primers are 5-TGGAATCCTGTGG CATCCA-3 and -TAACAGTCCGCCTAGAA GCA-3 (Integrated DNA Technologies). Cell proliferation assay CH27 B-cell lymphoma (1105 cells/ml) or splenic B cells (5105 cells/ml) from BALB/c or C57BL/6 mice were treated for 66?h with varying concentrations of CpG ODN, control GpC ODN, LPS (Sigma-Aldrich), the NF-B inhibitor 6-amino-4-(4-phenoxyphenylethyl amino)quinazoline,28,29 phorbol-12-myristate-13-acetate (PMA), ionomycin, or PMA plus ionomycin (EMD Chemicals, Billerica, MA, USA) in the presence of CpG or GpC ODN (7?g/ml). [3H]-thymidine (1?Ci; MP Biomedicals, Irvine, CA, USA) was added to each well during the last 18?h of incubation. Cells were harvested, and cell-associated radioactivity was measured using a scintillation counter. Apoptosis assay CH27 B-cell lymphoma cells (1105 cells/ml) and splenic B cells (4105 cells/ml) were incubated with or without 1 or 10?g/ml GpC or CpG ODN for 24 or 48?h. Apoptotic and necrotic cells were stained using an apoptosis detection kit (Invitrogen), as recommended by the manufacturer, and analyzed using a circulation cytometer (FACSCanto; BD Bioscience, San Jose, CA, USA). To neutralize Fas ligand, cells were incubated with anti-Fas ligand mAb (10?g/ml) (MFL4; BioLegend, San Diego, CA, USA) or an isotype control antibody (Armenian Hamster IgG; BioLegend) in the presence of 1 or 10?g/ml GpC or CpG ODN for 48?h followed by apoptosis analysis. TLR9 transfection TLR9 unfavorable CH27 cells were transfected with pUNO-mTLR9 (InvivoGen, San Diego, CA, USA) by electroporation using a Nucleofection kit (Lonza, Walkersville, MD, USA). After 24?h, the cells were incubated with 1 or 10?g/ml CpG ODN for 48?h and stained with Alexa Fluor 488-labeled Annexin V (Invitrogen). After fixation and permeabilization, cells were stained with an anti-mouse TLR9 antibody (IMAGENEX, San Diego, CA, USA) and analyzed using a circulation cytometer. Surface expression of Fas and Fas ligand by circulation cytometry CH27 cells (1105 cells/ml) were incubated with medium alone or 10?g/ml ODNs at 37 C for 48?h and were stained with anti-mouse FAS (CD95) antibody (BD Bioscience) plus an Alexa Fluor 405 conjugated secondary antibody (Invitrogen) or a PE-conjugated anti-mouse Fas ligand (CD178) antibody (BD Bioscience). Assessing NF-B translocation.