Fig 1: Activation of the NF-κB/AKT/β-catenin cascade by cNrf2-induced PSMD4 expression may be responsible for 5-FU resistance due to increased ZEB1 expression(A) The NLS-mutated Nrf2 plasmid was transfected into the shNrf2-HCT116 clone. After 24 h, the cells were treated with a proteasome inhibitor (carfilzomib), NF-κB inhibitor (BAY11-7082, BAY), AKT inhibitor (LY294002, LY), or β-catenin inhibitor (XAV939), and the cells were treated with six concentrations of 5-FU to calculate the IC50 value from the cell survival curves using the MTT assay. (B) NLS-mutated Nrf2 plasmid and/or shZEB1 were transfected into the shNrf2-HCT116 clone. After 24 h, cells were treated with six concentrations of 5-FU to calculate IC50 value by the MTT assay. (C) Western blotting analysis was performed to evaluate the expression of PSMD4, ZEB1, E-cadherin, vimentin, fibronectin, YAP1, and HO-1 following these treatments. The cells were treated with 0.1% DMSO or 10μM 5-FU for 24 h. The cells were subjected to annexin V and PI staining and flow cytometry analysis. The percentage of apoptotic cells, including the Annexin V+/ PI- population (early apoptosis) plus Annexin V+/PI– (late apoptosis/secondary necrosis), was determined by a flow cytometry analysis. Data are expressed as means ± s.d. (n = 3). (D) The NLS-mutated Nrf2 plasmid was transfected into the shNrf2-HCT116 stable clone. After 24 h, the cells were treated with three concentrations of carfilzomib. Western blotting analysis was performed to evaluate the expression of PSMD4, ZEB1, E-cadherin, vimentin, fibronectin, YAP1, and HO-1 in the stable clone subjected to these treatments. (E) The shNrf2-HCT116 stable clone was transfected with shp65, Shβ-catenin, shZEB1, and/or NLS-mutated Nrf2 plasmid. Western blotting analysis was performed to evaluate the expression of PSMD4, ZEB1, E-cadherin, vimentin, fibronectin, YAP1, and HO-1 in this clone subjected to these treatments. The cells were subjected to annexin V and PI staining and flow cytometry analysis. The percentage of apoptotic cells including the Annexin V+/ PI- population (early apoptosis) plus Annexin V+/PI– (late apoptosis/secondary necrosis) was determined by flow cytometry analysis. Data are expressed as means ± s.d. (n = 3).