Constitutive BLyS expression contributes to NHL-B cell survival in vitro. (A) XTT proliferation assay of NHL-B (LBCL-MS, MCL-Jeko) cells treated with control IgG or BLyS antibody. The number of live cells was monitored by the absorbance of 490 nm wavelength. (B) TUNEL analysis of NHL-B (LBCL-MS) cells treated with control IgG or BLyS antibody. Free DNA fragments in apoptotic cells were labeled with green fluorescence. (C) Proliferation of NHL-B (LBCL-MS) cells transfected with BLYS siRNA or control nontargeting sequence. DNA synthesis was assessed by [³H]thymidine uptake in vitro. (D) Immunoblot analysis of BLyS protein in NHL-B (LBCL-MS) cells transfected with BLYS siRNA or control siRNA. Actin was used as loading control. The BLyS level was analyzed by BioMax 1D software, normalized with actin levels, and presented as relative fold decrease compared to control samples. (E) TUNEL analysis of NHL-B (LBCL-MS) cells transfected with BLYS siRNA or a control siRNA. Free DNA fragments in apoptotic cells were labeled with green fluorescence. (F) Caspase-3 activity in an NHL-B (LBCL-MS) cell line transfected with BLYS or control siRNA. (G) Immunoblot analysis of Bcl-2 and Bcl-xL in NHL-B (LBCL-MS) cells transfected with BLYS or control siRNA. Bcl-2 and Bcl-xL levels were normalized to those of actin previously described in D. (H) Immunoblot analysis of BLyS, c-myc, and cyclin D1 in NHL-B cell line cells transfected with BLYS or control siRNA. Actin was used as loading control. The BLyS, c-myc, and cyclin D1 levels were normalized to those of actin as previously described in panel D. The data in panels A, C, and F are representative of 2 independent experiments. The error bars indicate standard deviation of triplicate samples.