NFATc1 or c-rel siRNAs inhibit CD154 promoter activity and cell growth in LBCL-MS cells. (A) LBCL-MS cells were cotransfected with the CD154-luc reporter construct (10 μg) with siRNA to c-rel or NFATc1 in a dose-dependent manner. After 48 hours, whole-cell extracts (50 μg) were analyzed by Western blot for NFATc1, c-rel, and actin. Actin was used for internal loading control. GFP siRNA was used as a negative control. (B) Whole-cell extracts from cells in panel A were measured for luciferase activity and normalized by β-gal activity. (C) LBCL-MS cells were transfected with siRNAs (100 nM) to GFP, c-rel, or NFATc1. Immediately after transfection, cells were processed for the proliferation assay described in “Materials and methods.” The data in part (B) and (C) are representative of 3 independent experiments. (D) Control and treated cells from panel C were also analyzed for apoptosis by TUNEL assay. Green staining represents apoptotic cells. (E) LBCL cell lines (McA, MZ, MS, and LP) were cultured in the presence of increasing doses of FK-506 (0-5 μg/mL) for 48 hours. DNA synthesis was assessed by [3H] thymidine uptake. The percentages of growth inhibition in treated cells were plotted with respect to untreated cells. The data shown are the means and ranges of triplicate cultures.