Responsiveness of NCM460 and RKO cells to Wnt stimulation. Wnt pathway throughput is measured following transient transfection of the luciferase-based SuperTopflash reporter construct. All experiments were performed in conjunction with Renilla luciferase determination to control for cell numbers, viability and transfection efficiency. All experiments were repeated at least three times and performed each time in triplicate. Panel A – Incubation with Wnt3a conditioned medium (CM) slightly, but significantly increased Wnt pathway throughput in NCM460 (p = 0.017) and very dramatically in RKO (p < 0.0001). Panel B – Exposure to Wnt2 producing cells in side-by-side (SbS) co-culture decreased Wnt pathway throughput in NCM460 cells (p = 0.027) but increased Wnt pathway throughput in RKO (p < 0.001). Panel C – Direct transfection of a Wnt2 expressin construct decreased Wnt pathway throughput in NCM460 (p < 0.0001) and increased Wnt pathway throughput in RKO (p < 0.0001). Panel D – In SbS co-culture with either Wnt1 or Wnt5a producing cells, Wnt pathway throughput was decreased by Wnt1 (p = 0.002) but unchanged by Wnt5a in NCM460 cells, and increased by Wnt1 (p < 0.0001) but unchanged by Wnt5a in RKO cells.