BR3 receptor activation promotes DLBCL cell growth through NIK protein stabilization. (A) Cell lysates from representative GCB-like (MS) or ABC-like (HB) DLBCL cells transfected with NIK shRNA or negative control (NC) shRNA for 48 hours were analyzed by Western blot analysis for NIK, p52, and pIkBα. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (B) Nuclear extracts purified from GCB-like (MS) or ABC-like (HB) DLBCL cells transfected with NIK shRNA or negative control (NC) shRNA for 48 hours were analyzed by DNA-binding ELISA for p52 and p65. (C) Proliferation of DLBCL cells (GCB-MS and SUDHL-4; ABC-HB and OCI-Ly3) transfected with NIK shRNA or negative control non-targeting sequence was analyzed by [3H] thymidine incorporation assays in vitro for 72 hours. The data shown are the means and ranges of triplicate samples of 2 independent experiments. Error bars represent SD. (D) Cell lysates from a representative GCB-like (MS) or ABC-like (HB) DLBCL cells transfected with BR3 shRNA or negative control (NC) shRNA for 48 hours were probed with BR3, NIK, pIκBα, p52, or caspase 3 antibodies by Western blot analysis. Actin was used as a loading control. Relative protein level of each target molecule was measured with ImageJ densitometer software and normalized to the actin level. (E). DLBCL cells (GCB-MS and SUDHL-4; ABC-HB and OCI-Ly3) were transfected with BR3 shRNA or negative control non-targeting sequence and cell proliferation was analyzed by [3H]-thymidine incorporation assays in vitro for 72 hours. The data shown are the means and ranges of triplicate samples relative to control samples of 2 independent experiments. Error bars represent SD.