Luciferase reporter assay kit

The 5′-flanking region of Zfp423 (−1324 to −764) was amplified by PCR and cloned into pCpGfree-Lucia (InvivoGen) luciferase reporter vector. Amplification of the reporter construct was performed using Dam⁻/Dcm⁻E. coli cells. These cells were purchased from the New England Biolabs and are mycoplasma-free. The luciferase reporter vector was in vitro methylated by incubation with 1 unit/μg of M.SssI enzyme (which methylates all CpGs) or 1 unit/μg of HhaI (which methylates the cytosines of the sequence GCGC) and HpaII enzymes (which methylate the cytosines in the sequence CCGG) at 37°C for 1 h. Fully methylated, unmethylated and partially methylated Zfp423 reporter vectors were transfected in NIH-3T3 cells. To normalise luciferase activity, a control plasmid encoding a Renilla luciferase gene was cotransfected into the cells. After 48 h, firefly and Renilla luciferase activity were assayed using a luciferase reporter assay kit (InvivoGen), according to the manufacturer’s instructions.

Media, sera, insulin, TRIzol and SuperScript III were obtained from Invitrogen (San Diego, CA, USA), rosiglitazone from Alexis (Grünberg, Germany) and 5-azacytidine, 3-isobutyl-1-methylxanthine and dexamethasone from Sigma-Aldrich (St Louis, MO, USA). pCpGfree-Lucia, Escherichia coli GT115 cells, and Luciferase reporter assay kit were from InvivoGen (San Diego, CA, USA), SYBR Green from Bio-Rad (Hercules, CA, USA) and the DNA Methylation Kit from Zymo Research (Orange, CA, USA). Micrococcal nuclease (MNase), Dam⁻/Dcm⁻Escherichia coli cells and HpyCH4IV, M.SssI, HhaI and HpaII enzymes were obtained from New England Biolabs (Ipswich, WI, USA). The DNA Purification Kit and pGEM-T Easy Vector were from Promega (Madison, WI, USA), the PCR Purification kit from Qiagen (Hilden, Germany), and the Big Dye Terminator v3.1Cycle Sequencing Kit from Applied Biosystems (Foster City, CA, USA).