Supertopflash

Cells were transfected with the respective siRNAs. After two days in culture, cells were further transfected with the SuperTopFlash reporter plasmid together with the internal control plasmid pGL4.74[hRluc/TK] (Promega) at the ratio of 100:1 and cultured for one day. Luciferase activities were measured by using Dual-Luciferase Reporter Assay System (Promega) and GloMax 96 Microplate Luminometer (Promega), according to the manufacturer’s instructions.

HEK293 cells (4 × 10
⁴/cm
²) were seeded in 12-well plates. 24 h later, 200 ng of SuperTOPFlash, 20 ng of Renilla luciferase (Promega, E2261), and 100 ng of GFP or PAWS1-GFP plasmids were co-transfected as described above. After 24 h, cells were treated with L-Wnt3A or L-conditioned medium for 6 h. Cells were washed with PBS and lysed in Passive Lysis Buffer (Promega, E194A). Firefly and Renilla luciferase activities were measured as described previously
^{17 (link)}. Briefly, extracts were mixed 1:1 with 2x Luciferase Buffer (50 mM Tris/phosphate (pH 7.8), 16 mM MgCl
₂, 2 mM DTT (dithiothreitol), 30% (v/v) glycerol, 1 mM ATP, 1% BSA, 0.25 mM luciferin and 8 μM sodium pyrophosphate) and light emission was measured using a Envision 2104 plate reader (PerkinElmer). An equivalent volume of 3x Renilla Assay Buffer (45 mM Na
₂EDTA, 30 mM sodium pyrophosphate, 1.425 M NaCl, 60 µM PTC124, 10 µM h-Coelenterazine) was then added, and Renilla luciferase emission was measured. The firefly luciferase counts were normalised to Renilla for each sample.