Figure 1. Co-expression of HER2CA and FLAG-NRF2 induces the expressions of NRF2-target proteins and increased the NRF2-dependent transcription in MCF7 cells.
(A) Overexpression of HER2CA increases the NRF2-induced expressions of HO-1 and MRP5 in MCF7 cells. MCF7 cells were transiently transfected with expression vectors by Lipofectamine PLUS as indicated and subjected to western blot analysis. β-actin was used as a loading control. The blots used in this experiment was cut into several pieces according to estimated M.W. of proteins of interest and probed indicated antibodies. Different pieces of the same protein blots or same samples with same conditions of electrophoresis and electrotransfer were used and presented. (B) HER2CA increases NRF2 DNA-binding in MCF7 cells. MCF7 cells were transfected with expression vectors as indicated and NRF2 DNA-binding assay was performed. As a control, MCF7 cells were treated with 100 µM of tBHQ for 8 hr. (C) HER2CA increases the ARE-Luc reporter gene activity in an NRF2-depedent manner. MCF7 cells were transfected with expression vectors as indicated with Lipofectamine PLUS and luciferase activity was determined as described in Materials and methods. (D) HER2CA increased transcriptional activation of HO-1 promoter by endogenous NRF2. MCF7 cells were transfected with HO-1 promoter-Luc and increasing amounts of HER2CA expression vector in the absence or presence of KEAP1 knockdown. Luciferase assay were performed as described in (C). (E) HER2CA increases the NRF2-dependent transcription of HO-1 promoter. MCF7 cells transfected with expression vectors by Lipofectamine PLUS as indicated in the presence of HO-1 promoter-Luc containing either wild type or mutant AP-1 site. Luciferase activity was determined as in (B). (B,C) Representative data were presented as mean ± SEM of triplicate experiments. Statistically significance was determined by two-tailed Student's t-test. *P ≤ 0.05; **P ≤ 0.01; and ***P ≤ 0.001.