Transcription reporter assays

HEK293T cells (ATCC) were routinely cultured in Dulbecco’s modified Eagle medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (Hyclone) and non-essential amino acids (Life Technologies). The cells in a well of a 6-well plate were transfected with 1 μg each of the protein-expressing plasmids and 0.5 μg luciferase reporter plasmid by Lipofectamine²⁰⁰⁰ (Life Technologies) following the manufacturer’s instructions. Three hours after transfection, the cells were replated into 96-well plates and were allowed to attach to the bottom of the plates during an overnight incubation. Then the cells were treated with different concentrations of 666-15 for 5–7 h. The luciferase activity was measured by a tube luminometer (Berthold) using luciferase assay reagent (Promega). The protein concentration of the cell lysates in each well was determined using a Protein Assay Dye Reagent (Biorad). The luciferase activity in each well was normalized to the protein content and expressed as RLuc (relative luciferase unit)/μg protein.