Luciferase reporter assay

Analysis on cancer-related pathways activity was performed using the Cignal Finder Pathway Reporter Arrays (SA Biosciences, Fredrick, MD). GBM cells were seeded into a 96-well white plate and incubated overnight at 37°C. Transient transfection was conducted by adding plasmid construct of transcription factor-responsive reporter of each pathway and controls to cells and incubated overnight in a 37°C incubator. Then, cells were further incubated for 48 hours. Each transfection condition was carried in triplicates. After 48 hours, the changes in expression of each pathway in cells with or without EMP3 depletion were determined by measuring the generated firefly and Renilla luminescent signals using the Dual-Glo Luciferase Assay system (Promega, Madison, WI) on the Glomax machine (Promega, USA). The relative luciferase units were determined by dividing the firefly to Renilla luciferase activity ratio. The luciferase activity of scramble shRNA control was regarded as 100%.