Transcription factor reporter assay

Cignal^™ 45-Pathway Reporter Arrays (Qiagen, CA) was used to screen for different transcription factors upon LPA stimulation of SKOV3.ip ovarian cancer cells. Cells were seeded into wells (50,000 cells/well) of the Cignal^™ Finder 96-well plates (Qiagen, CA) to transfect the reporters into cells via reverse transfection according to manufacturer's protocol. Briefly, reporter DNA constructs resident in each well of the plate were resuspended with 125 μl Opti-MEM and complexed with 25 μl of Lipofectamine 2000 (ThermoFisher, CA) transfection reagent. Each well is added with 5 × 10⁴ cells suspended 25 μl of Opti-MEM media. Transfection was allowed to happen by incubating the plate for 24 h at 5% CO₂ and 37°C. Following transfection, the cells were serum deprived for 16 h and treated with either vehicle (0.1% BSA in PBS) or LPA (20 μM) for 20 min. Differential activation of the transcription factors were determined by lysing the cells and measuring the luminescence intensity following the manufacturer's protocol.