Reduced TRAF6 protein expression in bortezomib-treated cells coincides with autophagy activation. (A) TRAF6 mRNA was measured by quantitative PCR in the indicated cell lines and CD34⁺ cells treated with increasing concentrations of bortezomib for 24 hours. (B) TRAF6 protein was measured by immunoblotting of the indicated cell lines treated with increasing concentrations of bortezomib for 24 hours. (C) HL60 and TF-1 cell lines cultured with bortezomib for 24 hours were evaluated by immunoblotting for LC3-I/II, p62, and GAPDH. (D) TF-1 cells were treated with the proteasome inhibitor, MG-132 (0, 1, and 10μM) for 24 hours and evaluated by immunoblotting for TRAF6, LC3-I/II, p62, and GAPDH. (E) TF-1 cells were treated with an autophagy inducer (100nM rapamycin) and an ER stress inducer (100nM thapsigargin). (F) Cell viability of THP-1 cells that have acquired resistance to bortezomib (Bort-R) and that are sensitive (Bort-S) was determined after treatment with increasing concentrations of bortezomib for 24 hours. (G) TRAF6 protein was measured by immunoblotting Bort-S and Bort-R cells treated with increasing concentrations of bortezomib for 24 hours. Values below the gel represent the intensity of TRAF6 relative to ACTIN.