Chromatin Immunoprecipitation and qPCR Analysis

Chromatin immunoprecipitation (ChIP)-PCR was performed as previously described [34 (link)]. In brief, 2 × 10⁶ OSCC cells were cross-linked with 1% formaldehyde and washed with PBS in the presence of protease inhibitors. Cells were homogenized and their chromatin was subjected to ChIP using magnetic Dynal beads (Invitrogen) and antibody against acetylated or trimethylated lysine 9 of histone H3 (H3K9) (Millipore, Temecula, CA). Fold-enrichment of amplified DNAs by ChIP was assessed using protocols as previously described [35 (link)]. Specific primers targeting the promoter region of TFPI-2 were as follows: R1-forward, 5′-GCAGGTCATTTCCGTCTAGCTT-3′ and R1-reverse, 5′-ACCTGCCTCCCAAACTTTCTC-3′; R2-forward, 5′-ACCACTTTCCCTCTCTTTTGCT-3′ and R2-reverse, 5′-TCGTAGTAGTAACGGAGAAGTAGGGC-3′.