Effects of NF-κB and NFAT inhibitors on CD154 expression in LBCL cells. (A) LBCL-MS cells were transfected with CD154p-luc (10 μg) along with 1 μg of pCMV–β-gal. Transfected cells were left untreated or treated with PS-341 (25 nM), BAY-11 (1 μM), FK-506 (5 μg/mL), CsA (5 μg/mL), TSA (5 μg/mL), or SAHA (5 μM). After 24 hours, luciferase activity was determined and corrected for transfection efficiency using β-gal activity. The data are representative of 3 independent experiments. The error bars indicate the standard deviation of triplicate samples. LBCL-MS cells were treated with different concentrations of PS-341, FK-506, or BAY-11 for 24 hours. Total RNA was purified and used for RT-PCR to detect CD154 mRNA level (B) and whole-cell lysates were analyzed for CD154 protein level by Western blotting (C). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and actin were used as internal control for panels B and C, respectively. DMSO was used as a vehicle control for PS-341. Regulation of CD154 by NF-κB in LBCL cells. (D) Supershift analysis of NF-κB proteins binding to the CD40L-κB site (-1180 to -1165). LBCL-MS nuclear extracts were incubated with the CD40L-κB, AP-1 cold probe or antibody against p50, p52, p65, c-rel, and rel-B. CD40L-κB-p³²-labeled probes were added to the binding reaction mixtures and analyzed by EMSA. comp indicates competition. (E) LBCL-MS cells were cotransfected with the 6xNF-κB-CD40L reporter plasmid (10 μg) with the expression vector pCMV-IκBαM (10 μg) or pCMV-empty (10 μg). Cells were treated with PS-341 (25 nM), BAY-11 (1 μM), or TSA (5 μg/mL). After 24 hours, luciferase activity was determined and corrected for transfection efficiency using β-gal activity. The data are representative of 3 independent experiments.