Chromatin immunoprecipitation (ChIP) and quantitative (q) ChIP assay

ChIP and qChIP analyses were performed using an EZ-ChIP kit (EMD Millipore) according to the manufacturer's protocol. In brief, PC-3 and DU-145 cells were cultured to 80–100% confluence, and the chromatin was cross-linked by 1% formaldehyde at 37°C for 15 min. Subsequently, cross-linked chromatin was sonicated (1 sec on, 1 sec off; 3×20 times) at 4°C to generate 200–1,000 bp fragments. Next, 5 µg anti-immunoglobulin G (IgG) antibody (cat. no. ab171870; Abcam) or anti-ZNF24 (cat. no. ab176589; Abcam) were used to immunoprecipitate chromatin fragments at 4°C overnight. IgG antibody was used as the control. After purifying the antibody-interact DNA, RT-qPCR was conducted to analyze the precipitated chromatin DNA, as aforementioned. The primer sequences were as follows: Twist1: Forward, 5′-AAGGGATGGACCTGAAACGG-3′; and reverse, 5′-GGCAAACTGGAAGCAGCAAA-3′. The qPCR conditions were as follows: 5 min at 98°C, denaturation at 98°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 20 sec, performed for 32 cycles.