Effect of Smac-mimetic on NF-κB signaling in MMCLs with different genetic abnormalities of NF-κB pathway components. (A) Immunoblot of of MMCLs cultured for 16 hours in medium alone or in the presence of 50nM Smac-mimetic. Nuclear and cytosol extracts were prepared, and expression of the proteins indicated at the right was analyzed. NF-κB mutations in different MMCLs are indicated. (B) Activation of NF-κB target gene expression after incubation of cells with Smac-mimetic. The average expression of 3 target genes (TNFAIP3, NFKB2, and IL2RG) was detected by quantitative PCR in control cells and cells cultured for 16 hours in medium with 50nM Smac-mimetic. Four groups were compared: (1) 3 MMCLs with biallelic deletions of cIAP1/2, (2) 5 MMCLs with mutations in TRAF3, (3) one cell line with biallelic deletions of TRAF2, and (4) 3 MMCLs with low NF-κB index and no known mutations in NF-κB pathway. The ΔNF-κB(3.1) index was determined as the differences between treated and control samples. Data are mean ± SE (“Genes comprising the NF-κB index in myeloma”). (C) Immunoblot for TRAF2 coimmunoprecipitated with NIK in MMCLs. In some cases, the cells were incubated with MG132 for 3 hours to stabilize endogenous NIK. NIK was immunoprecipitated and loaded on the gel (IP:NIK), or cytosol extracts were directly loaded. Samples were analyzed with TRAF2 antibody. (D) Immunoblot for NIK in MMCLs cultured for 16 hours in medium alone or in the presence of 50nM Smac-mimetic or Smac-mimetic plus MG132. NF-κB mutations in different MMCLs are indicated. (E) Proposed mechanism of activation of the NF-κB pathways through NIK-protein in MM cells with different genetic abnormalities (top to bottom): WT, ΔcIAP1/2, ΔTRAF2, ΔTRAF3, and NIK lacking TRAF3-binding site.