iCRT-3 inhibits Wnt signaling in TNBC cells. (A) Wnt pathway is antagonized by iCRT-3. BT-549 cells were serum-starved for 24 hours, and treated with Wnt-3a (200 ng/ml) and/or iCRT-3 (25 μM) for 4 hours. Total RNA was prepared, and assessed for Axin2 expression using quantitative real-time RT-PCR. β-actin was used as normalization control. Data represent mean ± SEM of three independent experiments (*p < 0.05). (B) iCRT-3 significantly inhibits transcriptional activity of β-catenin in BT-549 cells. BT-549 cells were transiently transfected with TOP-FLASH firefly luciferase reporter vector and Renilla luciferase vector. After 24 hour-transfection, cells were treated with DMSO or 25 μM iCRT-3 for 48 hours. Luciferase activities were measured using Dual-Luciferase Reporter Assay System. The relative luciferase activity was calculated by firefly luciferase activity/Renilla luciferase activity. Data represent mean ± SEM of three independent experiments (**p < 0.01).