Research Paper Volume 11, Issue 24 pp 12600—12623

Genome-wide global identification of NRF2 binding sites in A549 non-small cell lung cancer cells by ChIP-Seq reveals NRF2 regulation of genes involved in focal adhesion pathways

Figure 5. Conservative ARE analysis and luciferase reporter analysis of the LAMC1 gene. (A) Comparative transcription factor binding site analysis of the LAMC1 gene across different mammalian species shows highly-conserved NRF2 AREs. (B) Left, siNRF2-C27 and siGFP-C5 cells subjected to ChIP analysis with anti-Nrf2. Right, the relative ability of NRF2 to bind to the ARE site (value of NRF2 in siGFP-C5 cells set at 1; PCR reactions were not saturated; results are from at least 3 separate experiments; HO-1 served as positive control; GAPDH served as negative control). (C) tBHQ increased 151-bp LAMC1 promoter (sequence at left; red indicates ARE sequences)–luciferase activity in MCF7 cells. The plasmid pGL3-LAMC1-151bp was transfected into MCF7 cells in combination with pRL-TK for 24 h. Dual luciferase activity was measured after treatment with tBHQ (20 μM) for 6 h. Control, DMSO treatment for the same plasmid was set at 1 (mean ± SD, n=3; **p <0.01).