HEK293 cells (obtained from National Infrastructure of Cell Line Resource, Beijing, China) were seeded in a 96-well plate at a density of 5,000 cells per well and transfected with the SMAD-binding element (SBE)-SEAP plasmid using Fugene HD transfection reagent (Promega Corp.), as described previously (19 (link)), and the pSEAP basic plasmid as a negative control (both from Clontech Laboratories, Inc., Mountainview, CA, USA) prior to treatment with TGF-β1 and baicalin/baicalein. The SEAP signal was quantified using the SensoLyte® p-nitrophenyl phosphate (pNPP) SEAP Reporter Gene assay kit (AnaSpec, Fremont, CA, USA) according to the manufacturer's protocol. Briefly, after treatment with baicalin/baicalein (20–80 µM) for 48 h, cell supernatant was collected and heated at 65°C for 30 min to inactivate non-specific alkaline phosphatase activity. The assay buffer was then added and the mixture was incubated for 5 min at room temperature. Subsequently, pNPP substrate buffer was added and the mixture was incubated for 1 h at room temperature. OD values were then measured at 405 nm.