Combination of sorafenib and MG149 enhances ER stress response. (A) The relative mRNA levels of CHOP, IRE1, and sXBP1 (splicing form of XBP1) were determined by qPCR analysis (n = 3). (B) The protein levels of IRE1α, phospho-eIF2α, and CHOP in cells were determined by western blots. (C) The expression level of LC3B was examined using western blot analysis. (D) The ratio of LC3B-II and LC3B-I was calculated from the densitometric analysis of Fig. 3C (n = 3). (E) Representative fluorescent images revealed increased autophagic flux in the combination treatment (reduced intensity of GFP). (F) The relative GFP-LC3/RFP ratio was quantified by analysis of the intensity of GFP and RFP signals from treated cells in Fig. 3E (n = 10). (G) In all HCC cells, augmentation of aggresomes in combination with sorafenib and MG149 was detected by a PROTEOSTATⓇ aggresome detection kit. (H) Increased intracellular ROS levels were found after combination treatment with sorafenib and MG149 compared with single drug treatment. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not statistically significant.
