Fig. 2

Doxorubicin blocks bortezomib-induced aggresome formation in U266B1 cells. A–C Characterization of the formation of Vimentin cages and the aggregation of ubiquitinated misfolded proteins during aggresome formation. U266B1 cells were treated with 6.25 nM bortezomib for 0–24 h and analyzed by immunofluorescence to observe Vimentin and Ubiquitin (A). The percentage of cells with different forms of Vimentin (B) or Ubiquitin (C) was quantified and plotted. D, E Kinetics of Ubiquitin changes in cells with blurred or small Vimentin cages. U266B1 cells treated with 6.25 nM Bortezomib for 0–24 h were analyzed by immunofluorescence to observe Vimentin and Ubiquitin. The distribution of Ubiquitin in cells with blurred Vimentin cages (D) or small Vimentin cages (E) was quantified and plotted. Ubiquitin distribution was classified as: A, dispersed Ubiquitin; B, separated Ubiquitin particles; C, closely packed Ubiquitin particles; D, compact, clustered Ubiquitin. F Bortezomib stimulates aggresome formation in U266B1 cells. Cells were treated with 6.25 nM Bortezomib for 0–24 h, and immunofluorescence was used to observe Vimentin and Ubiquitin. Aggresomes were defined as Ubiquitin in type C or D, enclosed by blurred or small Vimentin cages. G Doxorubicin inhibits Bortezomib-induced aggresome formation. U266B1 cells were treated with no compound, 6.25 nM Bortezomib, 1 μΜ Doxorubicin, or the combination for 18 h, followed by immunofluorescence analysis for Vimentin and Ubiquitin. The percentage of cells with aggresomes, as defined in (F), was quantified and plotted. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test: *p < 0.05, ***p < 0.001. All experiments were performed independently three times. Scale bar: 10 μm