Chromatin immunoprecipitation (ChIP) assays

ChIP assays were conducted using the EZ ChIP™ kit (EMD Millipore) according to the manufacturer’s instruction. Briefly, melanoma cells were fixed with 1% formaldehyde to obtain protein-DNA crosslinks. Then cells were washed, lysed and sonicated to acquire chromatin fragments of 200–1000 bp. Subsequently, the chromatin was immunoprecipitated with antibodies against EZH2 (Abcam), H3K27me3 (Millipore) or IgG (Millipore), and then further incubated with magnetic beads. Precipitated chromatin DNA was recovered and analyzed by qRT-PCR. The primer sequences for PDCD4 promoter region amplification are listed as follows: PDCD4, 5′-GGATAGGTTGATTATTTGGAGT-3′ (sense) and 5′-TTATTTTTATTTTCTTCTACCCAAT-3′ (anti-sense). ChIP data is presented as a percentage relative to input DNA.