Fig. 2. Identification of Nrf2 as a positive regulator of the HBXIP gene.

A The transcription factor profiling assay identified TFs whose activity was altered in MCF-7 cells in response to detachment. B HBXIP promoter activity was investigated in TF-overexpressing MCF-7 cells. pGL-HBXIP FL promoter (from nt -2156 to +357, +1 from the TSS) reporter constructs were separately co-transfected with SATB1, Nrf2 and Sox2 expression vectors. The western blot shows SATB1, Nrf2, and Sox2 expression in transfected MCF-7 cells. C HBXIP promoter activity was investigated in MCF-7 cells and MDA-MB-436 cells co-transfected with pGL-HBXIP FL promoter-reporter constructs and increasing concentrations of the pCMV-Nrf2 plasmid. Nrf2 expression was detected by western blotting using an anti-Flag antibody. D HBXIP promoter activity in MCF-7 and MDA-MB-436 cells with Nrf2 knockdown. Nrf2 knockdown by two siRNAs was confirmed by western blotting. E HBXIP promoter activity was compared between Nrf2-overexpressing HEK293 cells and control cells. Nrf2 expression was confirmed by western blotting using an anti-Flag antibody. F Nrf2^−/− MEFs, Nrf2^+/+ MEFs, and human Nrf2-rescued Nrf2^−/− MEFs transfected with the pGL-HBXIP FL promoter-reporter plasmid were exposed to 50 μM H₂O₂ for 24 h and subjected to a dual-luciferase reporter activity assay. The error bars indicate the ±SD values as assessed by Student’s t test. All experiments were performed at least three times.