Plasmid construction

The promoter sequence of MMP9 from −761 bp to the transcription start site shows the strongest transcriptional activity. The MMP9 promoter (780 bp) was amplified by PCR using Fast Pfu DNA polymerase (TransGen Biotech, Beijing, China) from HTR-8/SVneo cDNA before cloning into the pGL3-Basic vector. The MMP9 promoter was mutated from GCGC to TAAT using the Fast Mutagenesis System (TransGen Biotech). We constructed various plasmids based on WT of MMP9 promoter in the pGL3-Basic vector: all CpG sites methylated except −712 bp, which was demethylated by mutation (−712mut); all CpG sites methylated except −233 bp, which was demethylated by mutation (−233mut); and all CpG sites methylated except −712 bp and −233 bp site were demethylated by mutation (−712 and −233mut) (Fig. 3E). The plasmid of Tet2-CD and Tet2-HXD was generously donated by Prof. Chang (24). All the primers used in this study are listed in Table S2.