Fig. 4

Cotreatment with doxorubicin increases misfolded protein accumulation, enhances ER/Golgi stress responses and promotes apoptosis/necroptosis in bortezomib-treated cells. A Cotreatment with doxorubicin increases the level of ubiquitinated proteins in bortezomib-treated cells. U266B1 cells exposed to bortezomib or the combination of bortezomib and doxorubicin were analyzed for misfolded protein levels by Western blot using anti-ubiquitin antibodies. The relative protein levels were quantified and plotted with statistical significance. B Cotreatment with doxorubicin elevates the protein levels of factors that promote ER stress-dependent apoptosis in bortezomib-treated cells. U266B1 cells exposed to bortezomib or the combination were analyzed by Western blot for the levels of ER stress response regulators, including IRE1, pIRE-S724, GRP78, CHOP, ATF3, and ATF4. The relative protein levels were plotted with statistical significance. C Cotreatment with doxorubicin increases the protein levels of factors involved in Golgi stress-dependent apoptosis in bortezomib-treated cells. Western blot analysis was performed on U266B1 cells treated with bortezomib or the combination, focusing on Golgi stress response regulators such as ARF4, CREB3, ETS1, ETS2, and HSP47. The relative protein levels were quantified and plotted. D Cotreatment with doxorubicin enhances the levels of proapoptotic factors in Bortezomib-treated cells. U266B1 cells were analyzed by Western blot for proapoptotic factors such as Bad, pBad (S112), Bax, Bik, Bim, BID, Bak, and Puma. The relative protein levels were plotted with statistical significance. E Cotreatment with doxorubicin increases the levels of active caspases in bortezomib-treated cells. Western blot analysis was performed on U266B1 cells exposed to Bortezomib or the combination, measuring the levels of the proform and cleaved active forms of caspases 3, 6, 7, 9, and PARP. The relative protein levels were plotted with statistical significance. F Cotreatment with doxorubicin elevates the protein level of RIPK1, a key factor promoting necroptosis, in Bortezomib-treated cells. U266B1 cells were analyzed by Western blot for necroptosis regulators, including RIPK1, RIPK2, and RIPK3. The relative protein levels were quantified and plotted with statistical significance. Statistical significance was determined using t-tests, with *p < 0.05, **p < 0.01, and ***p < 0.001. All experiments were performed independently three times