Luciferase Assay for GLI1 Promoter Activity

A modification of a previously described luciferase assay was used [15 (link)]. In brief, interleukin (IL)-6-stimulated and -unstimulated MF fibrocytes were transfected with luciferase reporter constructs that contained GLI1 gene promoter fragments. Reporter constructs were generated using pGL4.17[luc2/Neo] vector (Promega, Madison, WI, USA) and primers listed in Supplementary Table S8. The pRL-SV40 vector producing Renilla luciferase was used as an internal control. The constructs were transfected into fibrocytes using a lipofectamine transfection medium and incubated for 24 h at 37 °C. Following transfection, the fibrocytes were incubated in 2% FBS/RPMI-1640 starvation medium (Gibco) for 1 h at 37 °C and IL-6 (20 ng/mL) was added. Detection of luciferase activity was performed using the Dual-Glo system (Promega, Madison, WI, USA) and luminescence levels were measured using Monolight 3010 luminometer (BD Biosciences). The relative luciferase activity was calculated by normalizing the firefly luminescence to the Renilla luminescence.