Fig. 4. OPN interacts with β-Catenin in ICC.

a Interaction between both exogenous and endogenous OPN and β-Catenin. CCLP1 and HEK293T cells were transfected with empty vector or Flag-OPN and subjected to Co-IP and IB with anti-Flag antibody. Interaction of endogenous OPN with β-Catenin was detected by IP using anti-β-Catenin antibody with IgG as control. b Co-localization of OPN (red) and β-Catenin (green) in CCLP1 cells by IF with anti-β-Catenin and anti-OPN antibody respectively. DAPI was used for nuclei staining. c OPN and β-Catenin expression were positively correlated in 180 ICC tumor tissues. d, e ICC cell lines were subjected to WB analysis. Overexpression of OPN in RBE, HCCC9810, and HuH-28 cells could obviously promote the protein level of β-Catenin, cyclin-D1, c-Myc, and Prox1 comparing to control (d). Knockdown of OPN in HuCCT1 and CCLP1 cells significantly inhibit the Wnt/β-Catenin pathway (e). f The qRT-PCR result of ICC cells with OPN overexpression (RBE) or knockdown (CCLP1). OPN modulated the mRNA expression of C-MYC, CYCLIN-D1, and PROX1, which are the target genes of β-Catenin. Scale bar = 20 μm