Assays to determine if pHippy can be used to inhibit endogenous genes. a, Renilla luciferase assays to determine if any of 5 unique pHippy constructs against Human LRP6 inhibit activity of a fusion of LRP6 to Renilla luciferase. 293T were co-transfected with the vectors shown and assayed for luciferase activity 48 hours later. Cells co-transfected with LRP6Rluc and empty pHippy were set to 100%. All experiments were normalized for transfection with PGL3 luciferase. The average normalized Renilla luciferase levels and standard deviation are shown for 3 experiments. b, Determination of whether inhibition of endogenous LRP6 inhibits Wnt3a activation of the Wnt/β-catenin signaling pathway. 293T cells were co-transfected with the vectors shown, Super(8X)Topflash (to measure Wnt/β-catenin-signaling), and a constitutive expression vector for Renilla luciferase. 24 hours after transfection the cells were treated with Wnt3a conditioned media for 24 hours and then assayed for luciferase activity. Untreated cells transfected with empty vector were set to 1 fold activation of Super(8X)Topflash, which corresponds to ~10,000 RLUs. Cells treated with Wnt3a induce about 100 fold-activation of luciferase as shown. All experiments were normalized for transfection efficiency with the Renilla luciferase expression vector. One representative experiment is shown.