Figure 1

Canonical Wnt signaling upregulates p68 expression. (a) Whole cell lysates (WCLs) were prepared from MCF7, MDA-MB 231, 4T1, H1299, HCT116 and HEK293T cells. β-catenin, transcription factor 4 (TCF4) and p68 protein levels were analysed by immunoblotting (IB). Densitometry values are given below the blots. (b, c) Breast cancer patient samples (E-cadherin⁺ and E-cadherin^-) were analysed by immunohistochemistry (IHC) using antibodies against β-catenin, TCF4 and p68, as well as E-cadherin, to differentiate the samples. H scores of these samples were determined. (d) Wnt3a-MCF7 and empty vector (EV)-MCF7 stable cells were immunostained with β-catenin (primary) and Alexa Fluor488 (secondary, Green) antibodies and observed under fluorescence microscope to see its localisation pattern. Images were captured along with DAPI-stained nuclei at 600X magnification (scale: 10 μm) (top). Cytoplasmic and nuclear extracts were prepared from Wnt3a-MCF7 and EV-MCF7 stable cells and were analysed for β-catenin by IB. Densitometry values are given below the blots (bottom). (e) WCLs of Wnt3a-MCF7 and EV-MCF7 stable cells were analysed for Axin-2, β-catenin, Cyclin D1 and p68 proteins by IB. Densitometry values are given below the blots. (f, g) MCF7, MDA-MB 231 and 4T1 cells were serum starved for 24 h before treatment with Wnt3a condition medium (Wnt3a-CM). WCLs and total RNAs were prepared from 24 h post-treated cells to check the expression of β-catenin, p68 and Cyclin D1 by IB and qRT-PCR.