Analyzing Wnt Signaling in A549 Cells

The TOP/FOP flash assay was performed based on the protocol [30 (link)]. A549 cells were seeded in 96-well plate. When confluent, cells were transfected with 100 ng/well of either TOP luciferase reporter plasmid or the negative control FOP plasmid using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Invitrogen, Carlsbad, US) in serum-free Opti-MEM^® medium (Life Technologies, Carlsbad, US). After 5 h transfection, cells were stimulated with either vehicle, WNT-5A (50 ng/mL), or WNT-5B (50 ng/mL) in Opti-MEM^® medium supplemented with 0.1% FBS. After stimulation, cells were lysed by the Bright-Glo™ Luciferase Assay System (Promega). Subsequently, the luciferase activity was measured using a Synergy HTX Multi-Mode Microplate Reader (BioTek, Winooski, US). Data were collected with Gen5 software (BioTek).