2.2. Luciferase reporter assay

To establish HaCaT (3TP-luc) stable cells, cells were seeded on six-well plates. Cells were allowed to adhere overnight and then transfected with the p3TP-luc (neo) expression plasmid using PEI reagent (Sigma Aldrich). Transfected cells were cultured for four weeks in the presence of G418 (500 µg/mL). Several single clones were isolated and measured luciferase activity. The clone showing response to TGF-β1 treatment was used for reporter assay. HaCaT (3TP-luc) stable cells were seeded at 2.5 × 10⁴ cells/well in 96-well plate and were allowed to adhere overnight. Cells were concomitantly treated with TGF-β1 (2 ng/mL) and indicated concentrations of ALK5 inhibitors in 0.2% FBS medium and incubated for 24 h at 37 °C in 5% CO₂. Cell lysates were prepared using Luciferase Assay System (Promega) according to the manufacturer’s instruction, and luminescence was measured by a luminometer, Micro Lumat Plus (Berthold, Germany).