ChIP and Luciferase Reporter Assays

NHEK cells were incubated in high glucose for 5 d. ChIP assays were performed using ChIP-IT kit (Active Motif, Carlsbad, CA) following the manufacturer’s instructions. To precipitate FOXO1, anti-FOXO1 antibody (Santa Cruz Biotechnology, Inc.) was used, and the quantitative real-time PCR of MMP9 promoters was performed. ChIP experiments were repeated three times with reproducible results. A single putative FOXO1 binding site was predicted by PROMO bio prediction software located at −784 to −774bp in the human MMP9 promoter (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). Luciferase activity was measured with a Dual Luciferase Reporter Assay kit (Promega) according to the manufacturer’s instructions. In brief, NHEK cells were co-transfected with MMP9 Luciferase reporter³² (provided by Young Han Lee, Institute of Biomedical Science and Technology, Konkuk University, Korea) together with pRL-TK luciferase control vector, FOXO1-AAA plasmid that is constitutively transported to the nucleus, or pcDNA3.1 control plasmid, scrambled siRNA, or FOXO1 siRNA. 48 hours after transfection, cells were lysed, and Firefly and Renilla luciferase activities were measured using Dual Luciferase Reporter Assay kit (Promega) according to the manufacturer’s instructions. Firefly luciferase activities were divided by Renilla activities to normalize for transfection efficiency. Experiments were performed three times with similar results.