Cell-Based Reporter Assay for TGFBRII Signaling

Cell culture, DNA transfection, and cell-based reporter assays were carried out using established protocols [3 (link),55 (link)]. HEK293T cells were selected in this study, as they are easy to transfect and have a high transfection efficiency. HEK293T cells were seeded in a half-area of a 96-well tissue-treated microtiter plate and incubated for 24 h at 37 °C. The cells were transfected with plasmids encoding the TGFBRII receptor gene (100 ng), the SBE-Luc reporter (100 ng), and the pJ7Lac-Z plasmid (50 ng) using GeneJammer transfection reagent (Stratagene, San Diego, CA, USA) following the manufacturer’s instructions. Cells were then treated with drugs at 0.001 µM, 0.01 µM, 1 µM, and 10 µM for 24 h. Twenty-four hours after the treatment, the cells were lysed with 1× Reporter Lysis Buffer (Promega, Madison, WI, USA). Measurement of the luciferase and b-galactosidase was carried out as described elsewhere [3 (link)].