Top flash fop flash

The BCL-2 reporter plasmid (Addgene #15381) containing the BCL-2 promoter region from ATG to −3934 was a kind gift from Linda Boxer. A luciferase reporter assay system (Promega, Madison, WI, USA) was used to determine the luciferase activity of the BCL-2 promoter. U87/TMZ and U251/TMZ cells were seeded in a 96-well plate (8 × 10³ cells/well) and transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, Burlington, MA, USA) and 20 ng pRL-TK vector expressing Renilla luciferase (Promega), following the recommended protocol using Lipofectamine 3000 (Thermo Scientific). Cells were harvested 48 h later for analysis using a dual-luciferase reporter assay system (Promega). Luciferase activity was measured using a PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer). Luciferase intensity was normalized to Renilla luciferase activity to adjust for transfection efficiency.

U2-OS Cells were inoculated in a 96-well (5×10³ cells/well) and were incubated for 24 h, and then transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, MA, USA), and 20 ng pRL-TK vector expressing Renilla luciferase (Promega, WI USA) following the recommended protocol using Lipofectamine 3000 (Thermo Scientific, MA, USA) for another 24 h. Subsequently, the assays were divided into four groups: resveratrol (0, 6 or 12 μg/ml resveratrol for 24 h), CHIR-99021 (inhibitor of GSK-3α/β, Selleck, TX, USA) + resveratrol (the cells were pretreated with 10 μm CHIR-99021 for 24 h to activate the Wnt/β-catenin signaling pathway); lentivirus infection (shCx43, NTC and Blank), and lentivirus infection + XAV939 [(inhibitor of β-catenin, Selleck, TX, USA), the cells were treated with 10 μm XAV939 for 24 h to suppress the Wnt/β-catenin signaling pathway]. The OD values of the TOP flash and the FOP flash were detected by a dual-luciferase reporter assay system (Promega, WI, USA) from cell lysates, and the TOP/FOP ratio reflected the activity of the Wnt/β-catenin signaling pathway.