Regulation of MMP-9 Promoter by SOX5

The luciferase constructs containing the 2-kb promoter of MMP-9 gene were previously described (11 (link)). The putative SOX5-binding site from −1,300 to −900 was deleted by using the Quick Change II site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, USA) as previously described (11 (link)). MH7A cells were seeded on 24-well plates at 2 × 10⁵ cells/well and transfected with Ad-SOX5 or Ad-EGFP for 48 h. After overexpression of SOX5, 1 µg of full-length promoter/luciferase fusion plasmid DNA or the plasmid that deletion of SOX5-binding site was transfected into MH7A by Lipofect-AMINE kit (Invitrogen), respectively. 100 ng of pRL-SV40 control vector (Renilla luciferase) was co-transfected as an internal control for transfection efficiency. Luciferase activity in cell lysates was measured after 24 h with a dual luciferase reporter assay (Promega).