Fig. 5

Compound integration of β-catenin in the adherens junction (AJ) complex in hepatocellular carcinoma (HCC). a Growth curve of triple knockout HCC (TKO HCC) cells upon E-cadherin knockdown (KD) with shEcad1–2 (n = 4). E-cadherin protein level is determined by immunoblot. Double arrowheads indicate two migrating forms of E-cadherin. b Immunofluorescence (IF) for E-cadherin (magenta), β-catenin (green), and 4′,6-diamidino-2-phenylindole (DAPI (blue) in TKO HCC cells expressing an empty vector (control (CTRL)) or shEcad2 (E-cadherin KD). Scale bar = 25 μm. c Mass spectrometry (MS) analysis identifies the interactome of β-catenin in TKO HCC cells upon E-cadherin KD (shEcad2), relative to the control (CTRL). d Immunoprecipitation (IP) of immunoglobulin G (IgG) and β-catenin in TKO cells expressing an empty vector (CTRL), shEcad2 (E-cadherin KD), shNcad1 (N-cadherin KD), or shEcad2 + shNcad1 (E/N-cadherin double knockdown (DKD)). Endogenous β-catenin was pulled down and β-catenin, E-cadherin, N-catenin, and K-cadherin were detected by immunoblot. Double arrowheads indicate two migrating forms of E-cadherin. e β-Catenin, E-cadherin and N-cadherin expression in TKO HCC cells expressing an empty vector (CTRL), shEcad2 (E-cadherin KD), shNcad1 (N-cadherin KD), or shEcad2 + shNcad1 (E/N-cadherin DKD). Values in the lower panel represent the intensity of corresponding bands, normalized by actin. f IF for β-catenin (green) and DAPI (blue) in TKO HCC cells expressing an empty vector (CTRL), shNcad1 (N-cadherin KD), or shEcad2 + shNcad1 (E/N-cadherin DKD). g Growth curve of TKO HCC cells expressing an empty vector (CTRL) or shEcad2 + shNcad1 (E/N-cadherin DKD). (n = 3). Scale bar = 25 μm. h Subcutaneous tumors from nonobese diabetic/severe combined immunodeficiency (nod/scid) mice injected with TKO HCC cells expressing an empty vector (CTRL), shEcad2 (E-cadherin KD), shNcad1 (N-cadherin KD), or shEcad2 + shNcad1 (E/N-cadherin DKD). i Immunohistochemistry (IHC) for β-catenin in subcutaneous tumors. j Quantification of nuclei size in subcutaneous tumors from TKO HCC cells, control (CTRL) or E/N-cadherin DKD (n = 3). Scale bar = 200 μm, 60 μm (enlarged). k IHC for glutamine synthetase (GS) in subcutaneous tumor from TKO HCC cells with E/N-cadherin DKD. Cells with enlarged nuclei (arrowheads) do not express GS, indicating that Wnt activity is not required to trigger genomic instability. Scale bar = 50 μm. Data are represented as mean ± SEM. **p < 0.01 and ***p < 0.001. n.s. not significant. See also Supplementary Fig. 5