Figure 1.
Bortezomib-induced proteasome inhibition and autophagy.
(A) Bortezomib-induced proteasome inhibition. Proteasome activity was measured by fluorogenic assay after cells were treated with bortezomib (Millennium) for 16 hours. Error bars are omitted for clarity. (B) Bortezomib-induced cell death. Cell death was determined by flow cytometry of PI positive cells (PI+) after 24 hours treatment. Data presented (A and B) are mean ± SD from three independent experiments. Significant difference in cell death was observed by ANOVA with replicated data between DoHH2 and SU-DHL4 and Su-DHL10 (*P<0.0001) whereas no difference was observed between DoHH2 and Su-DHL8 (**P>0.05). (C) Bortezomib-induced accumulation of poly-ubiquitinated proteins and autophagy protein LC3-II. Cells were treated with bortezomib for 24 hours (DoHH2 and Su-DHL8) or 48 hours (Su-DHL4 and Su-DHL10). Cell pellets were lysed with 1% SDS containing lysis buffer. After heated at 99°C for 10 min, proteins were cleared by ultracentrifugation. Monoclonal anti-ubiquitin antibody was used at 1∶200 dilution. Polyclonal anti-LC3B antibody was used at 1∶1000 dilution and β-actin was used as a loading control. Numbers under each group of Western blots are ratios of ubiquitin/β-actin or LC3-II/β-actin which were measured by densitometry. (D) Autophagosome formation. After treatment with 20 nM bortezomib for 24 hours, fixed and permeabilized cells on slides were stained with the anti-LC3B antibody (1∶100 dilution) and then anti-rabbit Alexa Fluor-488 (1∶100 dilution). DAPI was used to indicate nuclear localization. Arrow heads indicate autophagosomes. (E) Formation of autophagosomes and autolysosomes. Cells were transfected with 1 µg pBABE-puro mCherry-EGFP-LC3B plasmids for 48 hours and then treated with 20 nM bortezomib for 24 hours.
Figure 2.
Bortezomib-induced cellular stress responses.
Bortezomib-induced ROS generation (A) and mitochondrial depolarization (B). Cells were treated with 20 nM (A) or different concentrations of bortezomib for 18 hours (DoHH2 and Su-DHL8) or 24 hours (Su-DHL4 and Su-DHL10). ROS or ΔΨm was determined by flow cytometry after cells were stained with HE or a ΔΨm-dependent dye TMRM, respectively. (A) A typical result from three independent experiments performed. (B) Data presented are mean ± SD from three independent experiments. By comparison with the DoHH2 cell line, significant differences by ANOVA were observed for Su-DHL4 and Su-DHL10 (*P<0.001), whereas no significant difference was observed for Su-DHL8 (**P>0.05). (C) Bortezomib-induced accumulation of CHOP was determined by Western blotting using anti-CHOP antibody at 1∶1000 dilution.
Figure 3.
Role of p62 on sequestration of ubiquitin and I-κBα.
Su-DHL8 cells were pre-incubated with or without 50 µM CQ for 1 hour and then treated with indicated concentration of bortezomib for 24 hours. (A) Co-localization of ubiquitin and p62. Cells on slides were co-stained with monoclonal anti-ubiquitin (1∶20 dilution) and polyclonal anti-p62 (1∶100 dilution) antibodies, and probed with Alexa Fluor anti-mouse IgG 546 (1∶50 dilution) and anti-rabbit IgG-488 (1∶100 dilution) antibodies. (B) Co-localization of p62 and LC3-II. Cells on slides were co-stained with monoclonal anti-p62 (1∶20 dilution) and polyclonal anti-LC3B (1∶100 dilution), and probed with Alexa Fluor anti-mouse IgG 546 (1∶50 dilution) and anti-rabbit IgG-488 (1∶100 dilution) antibodies. (C) Bortezomib-induced ubiquitination of I-κBα and accumulation of LC3-II protein. After treatment with 20 nM bortezomib with or without CQ, proteins were extracted from Su-DHL8 cells with 1% SDS and loaded onto 4–20% NuPAGE. Polyclonal anti-IκBα (1∶200) and monoclonal anti-ubiquitin (1∶200) were used to probe specific proteins. (D) Co-localization of p62 and I-κBα. Cells on slides were co-stained with polyclonal anti-I-κBα antibody (1∶20) and monoclonal anti-p62 antibody (1∶20) and then probed with Alexa Fluor anti-mouse IgG 546 (1∶50 dilution) and anti-rabbit IgG-488 (1∶100 dilution). Arrows indicate p62-I-κBα aggregates. (E) Detection of p62 and I-κBα interaction by Co-IP. Polyclonal anti-I-κBα antibody (2 µg) or normal rabbit IgG was coated onto Dynabeads protein A and 300 µg proteins were used for IP. Monoclonal anti-p62 antibody (1∶100 dilution) and polyclonal anti-I-κBα antibody were used to detect p62/I-κBα protein complex.
Figure 4.
Bortezomib-induced I-κBα phosphorylation and degradation.
DoHH2, Su-DHL4, Su-DHL8 and Su-DHL10 cell lines were pre-treated with 50 µM CQ for 1 hour and then treated with 10 nM or 20 nM bortezomib for 24 hours. Proteins were extracted in the presence of phosphatase inhibitor cocktails. Polyclonal anti-phospho-I-κBα (Ser32/36) (1∶1000) and anti-I-κBα (1∶200) antibodies were used for Western blotting. Ratios of I-κBα/β-actin (B) and P-I-κBα/β-actin (C) were analyzed by densitometry. Data represent mean ± SD from the four cell lines tested (*P<0.001). (D) Bortezomib-induced I-κBα degradation in primary lymphoma samples. Single suspension cells were treated with 20 nM bortezomib for 24 hours and proteins were extracted for Western blotting. Polyclonal anti-I-κBα antibody was used at 1∶200 dilution.
Figure 5.
Bortezomib-induced NF-κB activation.
Su-DHL8 cells were treated with 20 nM bortezomib with or without 50 µg/ml of CQ. (A and B) Effects of bortezomib or/and CQ on the levels of NF-κB proteins. Cytosolic and nuclear proteins were extracted from Su-DHL8 cells. 15 µg cytosolic or nuclear proteins were subjected to 4–20% NuPAGE gel. Anti-NF-κB RelA/p65, RelB, cRel, p50 and p52 rabbit antibodies were used at 1∶3000 dilution. Monoclonal Rb (the nuclear protein marker), and polyclonal LDH (the cytosolic protein marker) antibodies were used at 1∶2000 and 1∶500 dilution, respectively. Western blots presented are representative of 3 independent experiments performed. (B) Data analysis of NF-κB nuclear translocation. Significantly increased p65 or p50 protein levels after treatment with bortezomib (*P<0.05) was compared with their controls and analysed by t test (n = 3). Chloroquine-mediated inhibition on the nuclear translocation of RelA/p65, RelB, cRel and p50 was compared with their controls and treated with bortezomib alone. Significantly decreased nuclear protein levels were analyzed by t-test. (C) Effects of bortezomib or/and CQ on interaction between NF-κB and I-κBα. Dynabeads proteins A was coated with 2 µg of polyclonal anti-I-κBα antibody or normal rabbit IgG and then incubated with 300 µg protein. Interaction of I-κBα with NF-κB was determined by Western blotting with monoclonal anti-NF-κB antibody. IgG-Hv indicates IgG heavy chain (55 kD). Numbers under each pairs of bands are ratios of NF-κB/heavy chain. (D) Statistical data from three independent IP experiments analyzed by t test. (E) Effects of bortezomib or/and CQ on NF-κB RelA/65 DNA binding activity. Nuclear proteins were extracted using Nuclear Co-IP Kit. 10 µg nuclear proteins were used for the DNA binding assay. NF-κB DNA binding activity was determined using TransAM™ NF-κB p65 ELISA Kit. Data represent mean ± SD from 3 independent experiments. (F) Effects of bortezomib or/and CQ on NF-κB/cRel DNA binding activity. Su-DHL8 cells were treated with 20 nM bortezomib with or without 50 µM CQ for 6 hours. Protein-DNA complexes were extracted and bound with biotin-labeled NF-κB/cRel DNA binding site. Data represent two separate results from four independent experiments.
Figure 6.
Bortezomib-induced protein synthesis.
Su-DHL-8 cells were pre-treated with 50 µg/ml CHX for 1 hour and then incubated with 20 nM bortezomib for indicated period of time. (A) Effect of CHX on NF-κB and I-κBα. Monoclonal anti-NF-κB (1∶200) and anti-phospho-I-κBα (1∶1000), and polyclonal anti-phospho-NF-κB (1∶1000) and I-κBα were used for the Western blotting. (B) Effect of CHX on anti-apoptotic proteins. Polyclonal anti-Mcl-1 (1∶1000) and Bcl-XL (1∶200) antibodies, and monoclonal anti-Bcl-2 antibody (1∶200) were used for Western blotting. (C) Effect of CHX on other NF-κB target proteins. Goat anti-IL-6 (1∶1000) and rabbit anti-FLIP (1∶2000) antibodies were used for Western blotting. Numbers on the bottom of each lane are ratios of indicated protein to β-actin.
Figure 7.
Synergistic effect of CQ on bortezomib-induced cell death.
Cells were pre-treated with 50 µM CQ for I hour and then treated with bortezomib for 24 to 48 hours. (A) Determining cell death by flow cytometry. After treatment, cells were stained with 10 µg/ml PI and PI positive cells were counted as died cells. Data represent mean ± SD from 3 independent experiments. Asterisk (*) indicates P<0.01, significantly increased sensitivity analyzed by ANOVA. (B) Synergistic effect of CQ on bortezomib. Synergy was analyzed by CalcuSyn and synergy level was setup lower than 0.9. (C) PARP cleavage. PARP cleavage was analyzed by Western blotting using polyclonal anti-PARP antibody (1∶500). Lower bands (89 kD) indicate cleaved PARP. (D) Cell death in primary FL and DLBCL cells. Primary cells from liquid nitrogen were cultured in fresh medium for 3 hours. Cells were pre-treated with 30 µM CQ for 1 hour and then 20 nM bortezomib for 24 hours. Cell death was determined by flow cytometry. The significantly increased killing was analyzed by t-test.