Fig. 4

Differential Wnt axis activation upon esculetin treatment. A Relative gene expression profile of 84 canonical and non-canonical Wnt associated genes using RT2 profiler™ kit (Qigen corp). Cells were treated with esculetin for 24 h and were harvested to perform real-time PCR as per the manufacturer’s instructions. Untreated cells were taken as control. B Western blot analysis of β-catenin expression upon esculetin treatment. Total cell extract following 24 h of esculetin treatment was subjected to western blotting for β- catenin expression analysis. Representative blot of three independent experiments is shown. Densitometric analysis of relative β- catenin expression is shown in lower panel. (*p ≤ 0.05; **p ≤ 0.02). Full length immuno-blots are shown in supplementary data 1. C Intracellular calcium level analysis folowing 72 h esculetin treatment. Cells were harvested and stained with intracellular calcium binding dye -Fluo-4AM and analysed using flow cytometry. Relative fold change to untreated cells was plotted (lower panel). D Expression profiles of downstream targets of canonical and non-canonical axis using real time PCR following 48 h of 100 µM esculetin treatment. Data represents three independent experments (*p ≤ 0.05; **p ≤ 0.02). E Representative immunofluorescence images reveal a distinct distribution of β-catenin in esculetin treated/untreated cells (magnification 63X). Cells were fixed and stained with β-catenin antibody and FITC labelled secondary antibody. Representative images of three independent experiments are shown