TGF-β Signaling Pathway Activation Assay

SSCT fibroblasts were stably transfected with a reporter plasmid containing the Smad binding element. The Smad binding element comprises a CAGACA motif that serves as a direct binding site for Smad proteins using the Cignal Reporter Assay (Qiagen, Valencia, CA, CCS-017L) as the Smad binding element. Cells were plated at 1.3×10⁵ fibroblast cells per well and the following day cells were transfected with pathway reporters using FuGENE 6 transfection reagent (Promega Corporation, Madison, WI, E2691) according to the manufacturer’s protocols. After 24 hours culture, the transfected cells were pretreated with each inhibitor for 60 minutes and then treated with ± TGF-β (5ng/mL) as noted. After 24 hours cells were lysed with passive lysis buffer (Promega Corporation, Madison, WI, E1941) and cell lysates luciferase activity was measured upon addition of luciferase assay reagent (Promega Corporation, Madison, WI, E1483) using the Dual-Luciferase® Reporter Assay System (Promega Corporation, Madison, WI, E1910) as previously reported (Gingery et al., 2014 (link)). Relative units of luciferase activity were reported after subtracting the basal expression levels observed in the Veh group.