Applied biosystems simpliamp instrument

Rat MCCs were seeded in 10-cm² plates at a density of 1 × 10⁶ cells and stimulated with IL-6 (20 ng/ml) for 12 h. The cells were washed three times with ice-cold PBS. DNA and proteins were cross-linked by incubating cells with 1% formaldehyde for 10 min. Excess formaldehyde was quenched by incubating the cells with glycine for 5 min. The cells were lysed, and the nuclei were digested using micrococcal nuclease. Sheared chromatin was diluted and immunoprecipitated with 2 μg of an anti-ERRγ or control IgG antibody. DNA–protein complexes were eluted from Protein A/G agarose beads using a spin column, and the cross-links then were reversed by an incubation with NaCl at 65°C. ChIP assays were performed using a Chromatin Immunoprecipitation kit (Millipore). The relative binding of ERRγ to the ERRE regions of Mmp9 and Vegfa promoters was analyzed using PCR with an Applied Biosystems Simpliamp instrument (ThermoFisher, USA). The primers for ChIP assays were designed to amplify two different ERRE containing regions of the Mmp9 promoter and regions of the Vegfa promoter (Table S3). Agarose gel electrophoresis was used to detect DNA–protein binding and molecular weight of the precipitated DNA. For quantitative ChIP assays, RT-qPCR was performed using SYBR premixExTaq reagents (TaKaRa Bio) and an Applied Biosystems 7500 Real-Time PCR system (ThermoFisher, USA).

Chromatin immunoprecipitation (ChIP) assays were performed using the EZ-Magna ChIP A/G Assay kit (Millipore) according to the manufacturer’s instructions. Briefly, cells were crosslinked with 1% formaldehyde at 25 ± 5 °C room temperature for 10 min. Cells were then collected and lysed to isolate the nuclei with nuclear lysis buffer containing protease inhibitor cocktail (provided in the kit). Sonication was performed to shear chromatin, generating 200–1000 bp DNA fragments. The sheared chromatin was immunoprecipitated with primary antibodies, normal IgG, and anti-RNA polymerase II antibody (provided in the kit). After the protein-DNA crosslinking was reversed, the relative binding of EBF3 to the LINC01119 promoter was analyzed using PCR with an Applied Biosystems Simpliamp instrument (ThermoFisher, USA). Agarose gel electrophoresis was used to detect DNA–protein binding. Data were normalized to the input control.