Simultaneous inhibition of the proteasome activity and autophagy increases the CD20 levels in Raji cells. (A) Raji cells were incubated with indicated concentrations of bortezomib for 24 or 48 hours with or without 20nM bafilomycin A1 (Baf). Then, cells were stained with 1 μg/mL acridine orange for 15 minutes, washed with PBS, and analyzed in a FACSCalibur using CellQuest Pro Software Version 5.2. (B) Raji cells were seeded into the flask bottles at a concentration of 2 × 106 cells/10 mL. The cells were exposed to bortezomib at indicated concentrations for 24 or 48 hours with or without 20nM bafilomycin A1. CD20 was immunoprecipitated from samples with protein G bead slurry and analyzed with Western blotting. Blots were sequentially probed (after stripping) with antiubiquitin and anti-CD20 antibodies. IP indicates immunoprecipitation; and WB, Western blotting. (C) Raji cells were seeded into the flask bottles at a concentration of 2 × 106 cells/10 mL. The cells were incubated with bortezomib at indicated concentrations for 24 or 48 hours with or without 20nM bafilomycin A1. Then, cells were incubated with EZ-link sulfo-NHS-biotin. Labeled cells were lysed and biotinylated proteins were precipitated with immobilized NeutrAvidin protein. Subsequently, from the mixture of boiled precipitated surface proteins an additional immunoprecipitation step with anti-CD20 mAb (NCL-CD20-L26) was performed. Immunoprecipitates underwent electrophoresis followed by blotting with antiubiquitin, anti-CD20, antitubulin, or anti–ICAM-1 antibodies. L indicates the total cell lysate, positive control; and WB, Western blotting. (D) Raji cells were incubated with bortezomib at indicated concentrations for 24 or 48 hours with or without 20nM bafilomycin A1. Then, cells at a density of 105/100 μL were incubated with saturating amounts of FITC-conjugated mAb against CD20 for 30 minutes on ice in the dark and analyzed in a FACSCalibur using CellQuest Pro Software Version 5.2. *P < .05 (Student t test). (E) Raji cells were incubated with either diluent or bortezomib at 50-nM concentration for 24 hours or at 20-nM concentration for 48 hours with or without 20nM bafilomycin A1. Then, equal numbers of cells (105/well) were incubated for 1 hour with serial dilutions (from 1 to 100 μg/mL) of rituximab in the presence of 10% human AB serum as a complement source. Cell viability was measured with an MTT assay. The survival of cells is presented as percentage of corresponding diluent- or bortezomib-pretreated cells without rituximab. *P < .05 (Student t test).
