Functional consequences of 3q12.3/NFKBIZ copy gain and IκB-ζ overexpression. (A) CN of 3q12.3/NFKBIZ in 43 PTLs (left panel) and 49 PCNSLs (41 EBV^– and 8 EBV⁺, right panel) from the extension cohorts. Normals include 5 tonsils and 5 reactive lymph nodes. The upper 95% confidence interval of the normals was used as a threshold for copy gain. Indicated cell lines with known NFKBIZ CNs were used as controls. Cases with copy gain are shown in red. Error bars reflect standard deviation. (B) Cosegregation of genetic alterations in the TLR pathway (MYD88 mutations [upper panel; black, L265P; white, no L265P], NFKBIZ copy gain [middle panel; copy gain, red; color intensity corresponds to magnitude of copy gain]) and BCR pathway (CD79B mutations [lower panel; black, missense mutations affecting Y196; white, no exon 5 mutations; gray, not available]) in the 43 PTL samples (left panel), and 49 PCNSL cases (right panel; 41 EBV^– and 8 EBV⁺). (C) IκB-ζ encoded by NFKBIZ locus transcript abundance in representative DLBCL cell lines. Asterisks indicate cell lines with MYD88^L265P mutation. (D) IκB-ζ protein abundance in indicated cell lines. Full-length IκB-ζ is indicated with an arrowhead. Note that TMD8 has a heterozygous deletion of 159 base pairs, resulting in a shorter, fully functional IκB-ζ protein.³²  The membrane was reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. (E-F) Proliferation (E) and apoptosis (F) after knockdown of IκB-ζ (sh1 and sh2, 2 independent IκB-ζ hairpins; ev, control) in representative DLBCL cell lines with increased IκB-ζ transcript abundance resulting from NFKBIZ gain only (Ly4), MYD88 mutation only (TMD8), or both (HBL1). (G) Efficacy of knockdown of IκB-ζ and downstream targets was determined by immunoblot. (H) Proliferation after enforced expression of IκB-ζ in cell lines with low IκB-ζ transcript levels (DHL4 and K422). (I) Efficacy of IκB-ζ overexpression was determined by immunoblot.