W250d sonifier

A ChIP assay was performed using a Chromatin Immunoprecipitation Kit (Millipore, MN). Briefly, the cells were collected and crosslinked with 1% (v/v) formaldehyde for 10 min at room temperature. Nuclei were subsequently isolated via the lysis of the cytoplasmic fraction, and the chromatin was divided into 150–900 bp fragments via ultrasonic disruption using a standard microtip and a Branson W250D Sonifier (four pulses, 60% amplitude, duty cycle 40%). The sonicated nuclear fractions were divided for input control and incubated at 4 °C overnight with anti-Stat3 antibodies (Cell Signaling Technology, MA, USA) or control IgG antibodies (Cell Signaling Technology, MA). After incubating with 30 ml of ChIP grade protein A/G-agarose beads for 2 h at 4 °C, the antibody-protein-DNA complexes were eluted from the beads and digested using Proteinase K (40 mg) for 2 h at 65 °C, followed by spin column-based DNA purification. Finally, the genomic DNA recovered from the ChIP assays was amplified via qPCR with primers () specific for the Stat3 binding region of the CD147 promoter. The specificity of each primer set was verified by an analysis of the dissociation curve of each gene-specific PCR product.

Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP assay kit (Cell Signaling Technology) according to the manufacturer’s protocol. Briefly, 1 × 10⁷ cells were cross-linked with 1% formaldehyde for 10 min at 37 °C. Subsequently, chromatin was digested into fragments of 150–900 bp by micrococcal nuclease (400 gel units) for 20 min at 37 °C, followed by ultrasonic disruption of the nuclear membrane using a standard microtip and Branson W250D Sonifier (four pulses, 60% amplitude, duty cycle 40%). For immunoprecipitation, total chromatin was incubated overnight at 4 °C with 5 μg of the respective antibodies and rabbit IgG served as a negative control. The resulting precipitated DNA samples were analyzed by PCR using primers to amplify a potential binding site region of the ADAM17 promoter with specific primers. PCR products were resolved electrophoretically in a 2% agarose gel and visualized by ethidium bromide staining. The sequences of primers used for PCR are described in Supplementary Table 1. Each test was repeated in triplicate.