M50 super 8 topflash plasmid

The M50 Super 8× TOPFlash plasmid (#12456) and the M51 Super 8× FOPFlash plasmid (#12457) from Addgene (Watertown, MA). The HuH7 and 3H3 cells were seeded at a density of 1 × 10⁵ and 3 × 10⁴ cells per well in 24-well plates, respectively, and transiently transfected with 400 ng TOPFlash or FOPFlash plasmids and 100 ng pNL1.1.TK plasmid (Promega, Madison, WI) as an internal control by using PEI MAX (Polysciences, Warrington, PA). Two days after transfection, the relative light unit (RLU) was measured by using Nano-Glo Dual Luciferase Reporter Assay System (Promega) with FLUOstar OPTIMA-6 microplate reader (BMG Labtech, Durham, NC) following the manufacturer’s instructions. Luciferase activity was calculated as the ratio of firefly RLU (TOPFlash or FOPFlash) to NanoLuc RLU (pNL1.1.TK).

For assessing levels of Wnt signaling, 70% confluent cultures of NPCs in 24-well plates (with Neural Progenitor Medium; Stem Cell Technologies) were transfected with the M50 Super 8×TOPFlash plasmid (Addgene #12456; http://n2t.net/addgene:12456; RRID:Addgene_12456), which is used to assess β-catenin-mediated transcriptional activation. This plasmid contains a minimal TA viral promoter driving the expression of a firefly luciferase gene preceded by seven binding sites (AGATCAAAGG) for TCF/LEF^{67 (link)} (not to be confused with the TCF4 mutated in PTHS). Control NPCs were transfected with the M51 Super8×FOPflash plasmid, which has mutant TCF/LEF binding sites (Addgene plasmid #12457; http://n2t.net/addgene:12457; RRID:Addgene_12457).
Transfection was performed using the Amaxa Mouse Neural Stem Cell Nucleofector kit for NPCs (Lonza), using the manufacturer’s recommendations. After 24 h, medium was replenished and the luciferase assay was performed using the Pierce Firefly Luciferase Flash Assay Kit (Thermo Fisher Scientific) on a sample of 50,000 cells, using a Synergy microplate reader (BioTek Instruments). All assays were conducted on three independent replicates per NPC line (per subject) and three technical replicates. Activity levels were expressed as arbitrary units normalized against the mean activity in the respective controls.