Fig. 3
From: Promotion of growth factor signaling as a critical function of β-catenin during HCC progression
β-Catenin does not promote early hepatocellular carcinoma (HCC) through Wnt signaling. a Immunoblot for β-catenin in triple knockout (TKO) HCC cells and Wnt3A-inducible mouse fibroblasts used as controls for non-active vs. active Wnt signaling (L cells; parental or Wnt3a+) treated with 25 μg/ml cycloheximide (CHX) or dimethyl sulfoxide (DMSO) for the time indicated (0–24 h). b Comparison of ctnnb1 complementary DNA (cDNA) sequence in control liver (CTRL) and TKO HCC. Regions highlighted in blue correspond to the phosphorylation sites. c Immunoprecipitation (IP) of immunoglobulin G (IgG) and Axin in TKO HCC cells. The presence of Axin, glycogen synthase kinase 3β (GSK3α/β), and phospho-β-catenin in the pull-down fraction was determined by immunoblot. d TKO HCC cells and as L cells (parental or Wnt3a+) were treated with 5 μM XAV939 for 48 h. The expression levels of Axin and β-catenin were determined by immunoblot. e Axin cDNA was overexpressed in TKO HCC cells and L cells (parental or Wnt3a+). The expression levels of Axin and β-catenin were determined by immunoblot. f Immunoblot for β-catenin and glutamine synthetase (GS) in TKO HCC cells treated with Wnt3A media. g Immunohistochemistry (IHC) for GS in control liver (CTRL) and TKO HCC. Black arrowheads indicate central vein (CV). The tumor zone in TKO HCC is delineated by a white dotted line. Scale bar = 50 μm. h Quantitative reverse transcription PCR (RT-qPCR) analysis for glul, tbx3, lgr5, and axin2 in control liver (CTRL; n = 4) and TKO HCC (n = 9). i RT-qPCR analysis for glul, tbx3, lgr5, and axin2 in TKO HCC cells upon β-catenin knockdown (KD) with shβcat3-4 (n > 3). j Immunoblot for β-catenin and GS in TKO HCC cells overexpressing dominant-negative of TCF4 (dnTCF4). k RT-qPCR analysis for glul, tbx3, lgr5, and axin2 in TKO HCC cells upon dnTCF4 overexpression (n = 3). l Growth curve of TKO HCC cells upon dnTCF4 overexpression (n = 2). m Fold difference in the number of TKO HCC organoids upon dnTCF4 overexpression (n = 2). n Immunoblot of phospho β-catenin (Ser33/37/Thr41) and epithelial–mesenchymal transition (EMT) markers (Zeb1, Vimentin, and Claudin7) in control (CTRL) livers (n = 3), primary TKO HCC tumors (n = 4) and serially transplanted TKO HCC (Serial) tumors (n = 4). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. n.s. not significant. See also Supplementary Fig. 3
