Figure 4. Fisetin inhibits the EGF-induced transcriptional activity of MMP-9 by decreasing the DNA-binding activity of Sp1 on the matrix metallopeptidase-9 (MMP-9) promoter in ARPE-19 cells. ARPE-19 cells were treated with epidermal growth factor (EGF; 20 ng/ml) or were treated with EGF (20 ng/ml) plus fisetin (5 and 10 μM) for 24 h. A: Schematic of the transcription-factor binding regions on the human MMP-2 and MMP-9 promoter. B: Luciferase assay analysis of MMP-2 and MMP-9 transcription activity. C–E: Schematic representation of reporter plasmids of Sp1, NF-κB, and AP1 promoters (left to right) and responsive elements (upper portion). ARPE-19 cells were transiently transfected with reporter plasmids of Sp1, NF-κB, and AP1 promoters and were treated with fisetin for 2 h with or without EGF for 24 h. F: Schematic presentation of the MMP-9 promoter Sp1 mutant plasmid (shown at the top) and the luciferase activity of MMP-9. G: Chromatin immunoprecipitation (ChIP) analysis of the Sp1 binding to the MMP-9 promoter region treated with fisetin for 2 h with or without EGF for 24 h in ARPE-19 cells. Data are presented as the mean ± standard error of the mean (SEM) of at least three independent experiments. **p<0.01 compared with control cells; #p<0.01 compared with fisetin.