Canonical Wnt signalling is induced with human EC differentiation. NT2/D1 cells carrying a stably integrated multimerized canonical TCF-transcription factor binding site luciferase reporter were induced to differentiate. Luciferase activity was measured to determine the level of activity in the canonical Wnt signalling pathway and expressed relative to protein concentration. a) NT2/D1 cells transfected with siRNA for the transcription factor POU5F1 or control siRNA, and RA or vehicle control for 3, 7 and 9 days. b) Western validation of POU5F1 knockdown 3 days post siRNA transfection. POU5F1 levels of cells treated with RA were also measured after 3 days. c) Active-β-catenin levels in NT2/D1 cells following RA treatment. Lithium chloride (20 mM 24 hours) was used as a control to show activation of β-catenin, sodium chloride controlled for salt concentration. d) Time course of RA treatment over 7 days measuring integrated canonical Wnt reporter activity. Error bars represent standard deviation.