Figure 4

p68 co-activates β-catenin-mediated TCF4 gene expression. To examine this Wnt3a expressing stable MCF7 cells (wnt3a) were prepared and used where empty vector (EV)-MCF7 cells (ctrl) kept as control. (a, b) Stable MCF7 cells were serum starved for 48 h and analysed for expression of various transcription factor 4 (TCF4) target genes by immunoblotting (IB) and qRT-PCR. (c, d) p68 and β-catenin were either overexpressed (c) or knocked down using respective small interfering RNAs (siRNAs) (d) in Wnt3a-MCF7 cells and the expression pattern of p68, TCF4 and c-Myc were examined. Densitometry values are given below the blots. (e) Expression of p68, TCF4 and c-Myc was determined in EV-MCF7, Wnt3a-MCF7 and p68 knocked down Wnt3a-MCF7 cells by qRT-PCR. (f) Wnt3a-MCF7 cells were transfected with GFP-p68 (p68; green) and immunostained with anti-TCF4 antibody (primary) and Alexa Fluor 594 (secondary; red) and visualised under fluorescence microscope. Images were captured at 600X magnification (scale: 10 μm). (g) Tcf4 promoter activity was monitored in Wnt3a-MCF7 cells after overexpression of either β-catenin or p68 by transient transfection using luciferase assay.