Luciferase reporter assay

HEK293T cells were transduced with lentivirus coding for the pBARls reporter (Biechele & Moon, 2008) and with Renilla Luciferase as a control to generate a Wnt‐βcatenin signaling reporter cell line. To measure F4L5.13 activation, we transduced this cell line with lentivirus coding for human FZD4 protein. For luciferase assay, 2 × 10⁵ cells were seeded in each well of 24‐well plates for 24 h prior to stimulation. The following day, F4L5.13 protein was added, and following 15–20 h of stimulation, cells were lysed and luminescence was measured in accordance with the dualluciferase protocol (Promega) using an Envision plate reader (PerkinElmer). For TSPAN12 dependency, HEK293T‐TopFlash cells were seeded at 0.5 × 10⁶ cells in a 6‐well plate and the next day cells were transfected with FZD4, LRP5 and with either GFP or TSPAN12. Cells were transfected using Lipofectamine 2000 according to the manufacturer’s instructions. The next day, cells were passed in a 96‐well plate with around 35,000 cells/well, and after 5 h, cells were treated with either F4L5.13 or recombinant NDP overnight. After 16 h, cells were lysed, and luminescence was measured.