Levels of TRAF6 expression affect the cytotoxic effects of bortezomib. (A) THP1 cells were transduced with a bicistronic lentiviral vector encoding TRAF6 and green fluorescent protein (GFP) or an empty vector expressing only GFP. Transduced cells were sorted for GFP expression, cultured with bortezomib (10nM) for 24 hours, and protein lysates were evaluated for TRAF6 expression. (B) Cell viability of bortezomib-treated THP1 cells transduced with vector or TRAF6 was measured by annexin V/propidium iodide staining. (C) Summary of replicate experiments from panel B. (D) Bort-S and Bort-R THP1 cells were transduced with shRNA lentiviral vectors targeting TRAF6 or shCTL. Knockdown of the TRAF6 protein was confirmed by immunoblot analysis. (E-F) Cell viability of Bort-S (E) and Bort-R (F) cells transduced with shTRAF6 or control vector was determined by MTT assay after treatment with increasing concentrations of bortezomib for 24 hours (P < .05). (G-H) Proteasome inhibition in bortezomib-treated Bort-S (G) and Bort-R (H) cells transduced with shTRAF6 or control vector was determined by immunoblotting for ubiquitinated proteins (Ub) on total cell lysates. #P < .1; *P < .05.