Luciferase reporter assay

Cells were transfected with 50 nM GLI1 siRNAs or control siRNA. After 24 hours cells were co-transfected with the reporter plasmid ERE-TK-Luc and the pRL-TK control plasmid, which contains the Renilla luciferase gene, for normalizing transfection efficiency. Plasmid transfection was done using Lipofectamine 3000 (Invitrogen). After 24-hour plasmid transfection, the cells were changed to serum-deprived medium, incubated overnight, and then treated with 10 nM E2 or vehicle for 24 hours prior to harvesting. Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega). The reporter plasmid ERE-TK-Luc has been described previously [56]. Multiple t-test analysis (corrected for multiple comparisons using the Holm-Sidak method) was performed with GraphPad Prism 6.0d.