Chromatin immunoprecipitation kit

ChIP assay was performed using Chromatin Immuno-precipitation Kit (#17-10085, EMD Millipore, Billerica, MA, USA) according to the protocol of the Magna ChIP^TM A/G. For each sample, 1 × 10⁷ cells were fixed in 1% freshly prepared formaldehyde for 10 min at room temperature and glycine (125 mM) was added to quench unreacted formaldehyde for 5 min. Cells were then washed twice with ice-cold phosphate-buffered saline (PBS, pH 7.2) and lysed in cell lysis buffer (5 mM PIPES, pH 8.0; 85 mM KCl; 0.5% NP-40) for 15 min on ice. After centrifugation, the cell pellet was resuspended in nuclear lysis buffer (50 mM Tris-Cl, pH 8.1; 10 mM EDTA; 1% SDS). Cross-linked DNA was sheared with a Misonix Sonicator^® 3000 (Qsonica, Newtown, CT, USA) in six 15 s pulses with 50 s rest between pulses and a power setting of 6. Sheared chromatin was separated from debris by centrifugation at 10,000 × g for 10 min at 4°C. A total of 1 × 10⁶ cell equivalents of chromatin was used for immunoprecipitation. Chromatin was well mixed with 1 μg of the indicated antibody and protein A/G magnetic beads and incubated for 4 hours at 4°C with rotation. The chromatin-antibody-protein A/G complex was separated with a magnetic separator and washed. The complex was then digested with proteinase K at 62°C for 2 hours and beads were separated with a magnetic separation device. DNA was purified with a spin column for PCR.

Human colon cancer cell lines HT-29 and SW-620 were purchased from the Cell Center of Xiangya School of Medicine, Central South University (Hunan, China). Human normal colon cell line CCD-18Co was purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Both miR-133b mimics and inhibitor were synthesised by Shanghai GenePharma Co., Ltd (Shanghai, China). The transfection reagent Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA). P63 rabbit monoclonal antibodies were purchased from Abcam (Cambridge, UK). RhoA rabbit polyclonal antibody, E-cadherin rabbit polyclonal antibody and vimentin rabbit polyclonal antibody were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). GAPDH rabbit polyclonal antibody was purchased from Proteintech Group, Inc. (Chicago, IL, USA). The Alexa Fluor 488 Goat Anti-Rabbit IgG used for immunofluorescence was purchased from Invitrogen. The Chromatin Immunoprecipitation Kit was purchased from EMD Millipore Corporation (Billerica, MA, USA). A transwell system (24 wells, 8 μm pore size with poly-carbonate membrane) was purchased from Corning Costar (Tewksbury, MA, USA). Matrigel was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Chromatin Immunoprecipitation Kit (17–295, Merck Millipore) was used according to the recommended protocol. The lysates were incubated with anti-STAT3 or IgG. Immunoprecipitated DNA was collected using Protein A beads and was purified after phenol extraction and was used for qRT-PCR. Five sets of primers, including two sets covering two STAT3-binding sites and three sets without STAT3-binding sites were designed.

ChIP assays were performed using the Chromatin Immunoprecipitation Kit (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions. The DNA isolated from the ChIP assay with p53 antibody or normal IgG (negative control), along with an input control, was amplified using PCR to determine p53-binding elements around the promoter region of the BZLF1 gene (Zp). ChIP primers used here were as follows: BZLF1 promoter (−221-+12), 5′-GCAAGGTGCAATGTTTAGTGAG-3′ (forward) and 5′-CCATGCATATTTCAACTGGGC-3′ (reverse); p21 promoter (p53 REs), 5′-CTGGACTGGGCACTCTTGTC-3′ (forward) and 5′-CTCCTACCATCCCCTTCCTC-3′ (reverse). p21 promoter was used as a positive control.