Figure 1.
b-AP15 treatment causes accumulation of high molecular weight polyubiquitinated proteins and induces apoptosis.
A. HCT116 cells were cultured in the presence of 1 µM b-AP15 or 100 nM bortezomib and harvested at the indicated time points. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, HSP-70B', p21, PARP or β-actin (loading control). B. HCT116 cells or hTERT-RPE1 cells were exposed to different concentrations of b-AP15 for 1 hour, followed by washing and incubation for 16 hours in drug-free medium. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, PARP and β-actin (loading control).
Figure 2.
CpdA inhibits co-translational translocation of endogenous prosaposin into the ER.
A. and B. HCT116 cells were treated for 1 h with DMSO, tunicamycin (10 µg/ml) or CAM741 (10 µM) before pulse-labelling for 10 min with [35S] Met/Cys. Endogenous prosaposin (pSAP) was recovered by immunoprecipitation and newly synthesised pSAP species were visualised by phosphorimaging. In DMSO-treated cells, pSAP was fully glycosylated (pSAP-5), whereas Endo H digestion or tunicamycin treatment yielded non-glycosylated pSAP (pSAP-0). Inhibition of protein translocation into the ER by CAM741 resulted in the appearance of a pSAP species that migrated more slowly than the non-glycosylated protein and was Endo H-resistant. This species may represent signal sequence-containing preprosaposin (prepSAP-0) that has failed to translocate across the ER membrane. C. Distinct forms of endogenous pSAP in HCT116 cells treated with DMSO, b-AP15 (1 µM), bortezomib (20 nM), cpdA (10 µM) or tunicamycin (10 µg/ml) were recovered by immunoprecipitation and visualised by phosphorimaging. Treatment with cpdA specifically inhibits the co-translational translocation of pSAP into the ER as judged by the appearance of prepSAP-0 species.
Figure 3.
CpdA enhances the ability of b-AP15 to inhibit proteasomal degradation.
A. MelJuSo cells stably expressing UbG76V-YFP were treated with 1 µM b-AP15 for 6 hours or left untreated (control), and cell lysates subjected to immunoblotting using anti-YFP. The migration of UbG76V-YFP and the expected position of higher molecular weight polyubiquitinated forms (poly-Ub) are indicated. B. MelJuSo-UbG76V-YFP cells were exposed to 1 µM b-AP15 for 8 hours or left untreated (control). Cells were labeled with an anti-K48 polyubiquitin antibody followed by an allophycocyanin conjugated secondary antibody and analyzed by FACS. C. and D. MelJuSo-UbG76V-YFP cells were exposed to different concentrations of b-AP15 in the presence or absence of 10 µM cpdA as indicated. Changes in the number of fluorescence-positive cells/field following addition of the compounds were monitored using an IncuCyte-FLR microscope.
Figure 4.
b-AP15 treatment alters the subcellular distribution of ubiquitin and causes the appearance of ubiquitin positive inclusions.
HeLa-M cells transiently transfected with FLAG-ubiquitin were treated with 10 µM cpdA (top panels), 1.0 µM b-AP15 (middle panels) or 0.8 µM b-AP15 plus 10 µM cpdA (bottom panels) for 16 h. Cells were fixed and stained with anti-FLAG and anti-calnexin antibodies followed by fluorescently labelled secondary antibodies. Confocal images were collected and images show the combined optical stacks. Scale bar = 10 µM.
Figure 5.
CpdA increases cellular levels of polyubiquitinated proteins and sensitizes cells to b-AP15 induced cell death.
A. HCT116 cells were pre-treated with or without (control) 10 µM cpdA for 16 hours, exposed to the indicated concentration of b-AP15 for 1 hour, then incubated for a further 16 hours in drug-free medium. Lysates were subjected to immunoblotting for K48-linked polyubiquitin, PARP and β-actin (loading control). B. HCT116 cells were treated as above. Following treatment, the number of dead cells was measured by Trypan-blue staining. Bar chart shows mean +/− SD of three independent experiments. Statistical significance was calculated using the Student's t-test. P values * = 0.05 and ** = 0.01. C. and D. hTERT-RPE1 cells were treated and analyzed as above. E. and F. Human diploid fibroblasts were treated and analyzed as above, and the number of dead cells was determined 36 hours after drug treatment.
Figure 6.
Cysteine supplementation does not protect HCT116 cells from b-AP15 toxicity.
A. HCT116 cells were exposed to 1 µM b-AP15 or 100 nM bortezomib (BZ) for 8 hours or 16 hours, in the presence or absence of 1 mM cysteine as indicated. Cell lysates were subjected to immunoblotting for LC3-I/II and β-actin (loading control). B. HCT116 cells were exposed to 1 µM b-AP15 for 16 hours in the presence or absence of 1 mM cysteine. Where indicated, cells were pre-treated with 10 µM cpdA for 16 hours prior to b-AP15 treatment. Apoptosis was determined by the appearance of caspase-cleaved K18. Bar chart shows mean values ± SD of of three independent experiments.
Table 1.
Treatment of HCT116 cells with b-AP15 and cpdA does not deplete cellular cysteine levels.