Dual-Luciferase Reporter Assays

A dual-luciferase reporter assay was performed as previously described (Zhan et al., 2014; Kong et al., 2015). Shh-LIGHT2 cells, an Hh activity reporter cell line derived from NIH-3T3 cells by stably expressing 8 × Gli-binding site luciferase reporter (8 × GBS-luciferase) plasmid provided by Sasaki et al. (1999) and pRL-Renilla luciferase plasmid, were seeded into 96-well plates. After 24 h, ShhN-conditioned medium (ShhN CM) was added, and cells were exposed to molecules. The luciferase activities of cells were measured 36 h later with a Dual-Luciferase Reporter Assay System (E1960, Promega), according to the manufacturer's instructions, in a luminometer (Molecular Devices, Sunnyvale, CA). The firefly luciferase values were normalized to the Renilla luciferase activities, and IC₅₀ values were fitted with the Hill equation with OriginPro 9.