BR myeloma cells remain sensitive to bortezomib treatment and recover proteasome activity. (A) The degree of resistance (D.O.R.) of cells tolerant of bortezomib, compared with their drug-naive counterparts, was evaluated by examining live cell populations using the WST-1 proliferation assay after exposure to bortezomib for 24 hours. Data shown are representative from triplicate experiments. (B) Apoptosis was evaluated in 8226.wt and 8226.BR cells by staining with annexin-V and TO-PRO-3. Data shown are representative from duplicate experiments. (C) Dose-dependent effects of bortezomib on the proteasome ChT-L activity were measured using cellular extracts. Inset, ANBL-6.wt and ANBL-6.BR cells propagated in the absence of bortezomib were probed for their content of ubiquitin-protein conjugates. (D) Time-dependent effects of bortezomib (10nM) on proteasome ChT-L activity were examined in 8226.wt and 8226.BR cells. Inset, expression of the basal level of the β5 proteasome subunit was examined by Western blotting and quantified by densitometry after normalization for loading using β-actin as a control. Data are representative from experiments performed in triplicate, and error bars indicate SD.