2.6. Gli-Luciferase Assay

NIH3T3 cells and Ptch1^−/− mouse embryonic fibroblasts (MEFs) were used for this assay. Cells were grown in a 10 cm culture dish to an approximate confluence of 90%, trypsinised, and seeded in 24-well plates at a 1:5 dilution in growth media. Cells were transfected 24 h after plating using different transfection reagents for each cell line: FuGENE^®HD (Promega, Southampton, UK; cat. E2311) for NIH3T3 and TransIT-X2^® (Mirus Bio, Madison, WI, USA; cat MIR6003) for MEFs.

Assays were always conducted in triplicate. For each well, a fixed amount of p8xGBS-Luc (135 ng), pRL-SV40 or pRL-TK (15 ng/well) and testing plasmid DNA (375 ng, containing a single plasmid or a combination of plasmids) and 1.5 µL of the transfection reagent were used, following the manufacturer’s instructions. After 24 h, transfected cells were incubated with serum starvation media (DMEM, 0.5% FBS, 1% GlutaMAX) for an additional 48 h. Cells were washed with 0.5 mL of PBS and lysed with 100 µL passive lysis buffer with vigorous shaking at room temperature for 15 min. Dual-luciferase reporter assay system (Promega; cat. E1910) was used to measure the activity of Firefly-luciferase and Renilla-luciferase using a Promega GloMax 20/20 luminometer (cat. E5311). Values of the individual measurements were generated as relative luciferase units (RLUs) and as Firefly-luciferase/ Renilla-luciferase ratio.