Figure 4.

CHOP induction critically contributes to the dilation of the ER and subsequent cell death by bortezomib/nultin-3. (a) Cell extracts were prepared from MDA-MB 435S cells treated with the indicated concentrations of bortezomib and/or nutlin-3 for 8 h and western blotting of the proteins associated with ER stress was performed. β-actin was used as a loading control in western blots. (b) Cell extracts were prepared from MDA-MB 435S cells treated with 5 nM bortezomib plus 30 μM nutlin-3 for indicated time points and western blotting of ubiquitin and CHOP was performed. β-Actin was used as a loading control in western blots. (c–e) MDA-MB 435S cells were infected with the lentivirus containing non-targeting (NT) shRNA or a CHOP-targeting shRNA (CHOP shRNA) for 24 h. Infected cells were treated with 5 nM bortezomib plus 30 μM nutlin-3 for 24 h (c, d) or for 16 h (e). (c) Cell viability was assessed using calcein-AM and EthD-1. The percentage of live cells was normalized to that of cells transfected with shNT without treatment (100%). Data represent the means±s.d. One-way ANOVA and Bonferroni’s post hoc test. *P<0.005 vs cells transfected with shNT without treatment, ^#P<0.005 vs cells transfected with shNT and further treated with bortezomib/nutlin-3. The experiment was repeated at least three times with similar results. (d) Treated cells as indicated were observed under the phase-contrast microscope. Scale bar, 10 μm. (e) Treated cells as indicated were fixed and subjected for immunocytochemistry of PDI and COX II. Scale bars, 20 μm.