SHH Reporter Assays

Analysis of hedgehog signaling activity was performed with SHH-LIGHT2 cells, a clonal NIH-3T3 cell line stably expressing a Gli-dependent firefly luciferase and constitutive Renilla reporters, as previously described³¹ with minor modifications. SHH-LIGHT2 cells were seeded in 96-well plates at a density of 4000 cells per well in 200 µL of DMEM with 10% FBS. Once confluent, cells were cultured for a further 12 to 24 hours before treatment with conditioned media. Conditioned medium was prepared by transfecting HEK293 cells with SHH-GFP, HHIP-FLAG, HHIPL1-GFP, or GFP control plasmid. Twenty-four hours after transfection, medium was changed to DMEM with 0.5% FBS and collected after a further 24 hours for treatment of SHH-LIGHT2 cells. Firefly luciferase and Renilla were measured 24 hours after treatment using the Dual-Glo Luciferase assay system (Promega) and read on a Novostar plate reader (BMG LabTech). The hedgehog signaling activity of mouse AoSMCs was measured by co-culturing wild-type or Hhipl1^−/− cells with SHH-LIGHT2 cells. Three wild-type and 3 Hhipl^−/− AoSMCs were used for experiments. Cells were seeded at 5000 SHH-LIGHT2 and 2500 AoSMCS per well in 96-well plates in replicates of 3 to 6. When cells reached confluence, medium was changed to DMEM containing 0.5% FBS. Luciferase activity was measured 24 hours later.