Fig. 2.

NRF2 negatively regulates the expression of FOCAD. To downregulate the expression of NRF2, A549 cells were treated with brusatol (10 nM, 30 nM, 50 n M for 16 h) (A–B) or siRNA (10 nM for 48 h) (C–D). NRF2 knockout (KO) cell line was also established using CRISPR/Cas9 (C–D). Then, the cell samples were harvested for western blot (A and C) and qPCR detection (B and D). In addition, biotinylated DNA probes (41 bp) spanned the ARE containing sequences in the promoter regions of human FOCAD gene, and the DNA-protein complexes were pulled down using streptavidin beads for immunoblot detection (E). To confirm the model, RPA1 knockout cell line (A549-RPA1^−/−) was also established and pGL3-FOCAD-ARE plasmid and hRluc/TK plasmid were cotransfected into the cell using Lipofectamine 3000. 24 h later, the cells were treated with brusatol (50 nM for 16 h) or ML385 (5 μM for 16 h) for luciferase activity assay (F). Results were expressed as mean ± SD (n = 3), and the P value less than 0.05 was considered statistically significant. *: P < 0.05 compared with the control (without any treatment) group.