PRDM1 was expressed in T-cell lymphoma and could be down-regulated by bortezomib through NF-κB inactivation. (A) Nuclear PRDM1 protein expression was revealed by immunohistochemistry on lymphoma tissue. Leica AS LMD (Leica Microsystems), 40×/0.6 CORR XT, air/0.6, hematoxylin-eosin stain, Video camera 3CCD Hitachi HV-Dz0P, Leica AS LMD software, version 4.4. (B) PRDM1 mRNA and protein expression were detected by semiquantitative PCR on lymphoma cells and Western blot on lymphoma tissue, respectively. PRDM1 isoforms were expressed in laser-microdissected lymphoma cells and T- and B-lymphoma cell lines (NA indicates namalwa; SU, SU-DHL-4), but not in peripheral blood lymphocytes (PBLs). (C) Laser-microdissected lymphoma cells showed coexpression of PRDM1 and IRF4. (D) In HUT78 cells resistant to doxorubicin (Dox), PRDM1β, IRF4, and c-MYC expression was reduced by bortezomib (Bor) both at transcriptional (top panel) and protein (bottom panel) level. (E) In HUT102 cells resistant to 6-mercaptopurine (6MP, 50 μM), PRDM1β, IRF4, and c-MYC expression was also reduced by bortezomib (Bor, 25 nM) (top panel). In MOLT4 cells, when methotrexate resistance was developed (MTX, 150 nM), PRDM1β was expressed, and subsequently reduced by bortezomib (Bor, 25 nM) (bottom panel). (F) Immunofluorescence study revealed that NF-κB (P65) was located mainly in nucleus in doxorubicin (Dox)–treated HUT78 cells, while in cytoplasm in those treated with bortezomib (Bor). Leica AS LMD, 100×/1.3 oil, oil/1.3, Immunofluorescent stain, Video camera 3CCD Hitachi HV-Dz0P (Hitachi, Tokyo, Japan), Leica AS LMD software, version 4.4. (G) Bortezomib (Bor) blocked NF-κB nuclear translocation and retained the phosphorylated form of IκBα (p-IκBα) and IκBα in the cytoplasm of HUT78 cells. Lamin B was used as a nuclear protein control. (H and I) Addition of NF-κB inhibitor PDTC increased the antiproliferative effect of doxorubicin (Dox) on HUT78 cells (H) and induced PRDM1β down-regulation (I).