Fig 2: 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

Fig 3: HPSE contributes to β-catenin activation and signal transduction following HSV-1 infection. (A) Rationale for mechanistic investigation of role of β-catenin in HSV-1 infection. Using quantitative proteomics analysis of HSV-1 infection time course of wild-type and Hpse-KO MEFs, β-catenin (Ctnnb1) was identified as a major differentially expression hub gene. (B) Proposed mechanism for HPSE involvement in β-catenin signaling following HSV-1 infection. (C) Confocal immunofluorescence microscopy of β-catenin expression and localization in HCE cells transfected with constitutively active HPSE (GS3-HPSE) or empty vector (EV) and infected with HSV-1 K26-GFP at MOI of 0.1. (D) Significance between the corrected total nuclear fluorescence determined by an unpaired t test. (E) Confocal immunofluorescence microscopy of β-catenin expression and localization in WT and Hpse-KO MEFs infected with HSV-1 K26-GFP at MOI of 0.1. Arrows indicate cells where β-catenin expression is heightened and nuclear localization is observed. (F) Significance between the corrected total nuclear fluorescence determined by an unpaired t test. For all statistical analysis, *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

Fig 4: Forced expression of Sp5 promotes EpiSC reprogramming to naïve pluripotency. (A) Phase contrast and alkaline phosphatase (AP) staining images of 46C mESCs and 46C mESC-derived EpiSCs. Scale bar: 100 μm. (B) qRT-PCR analysis of expression patterns of Sp5, Oct4, Rex1 and Fgf5 in 46C mESCs and EpiSCs. Data represent mean±s.d. of three biological replicates. **P<0.01 versus 46C mESCs. (C) Western blot analysis for Sp5 in 46C EpiSCs transfected with vector expressing FLAG-tagged Sp5. (D) Phase contrast and alkaline phosphatase staining images of Sp5 EpiSC transfectants and Sp5 EpiSC transfectant-derived induced pluripotent stem cells (iPSCs). Scale bar: 100 μm. (E) qRT-PCR analysis of Oct4, Nanog, Stella, Nr0b1, Klf2, Rex1, Fgf5 and Sox17 expression in Sp5 EpiSCs and two Sp5 Epi-iPS colonies. Data represent mean±s.d. of three biological replicates. **P<0.01 versus Sp5 EpiSCs. C1 and C2 are the individual colonies of ESC-like cell. Epi-iPS, EpiSC-derived iPSCs.

Fig 5: FOXC1 enhances c-MYC expression to promote gastric cancer cell proliferation through activation of the Wnt signaling pathway. (A) TopFlash/FopFlash assays using normal gastric cells (GES-1) and gastric cancer cells (AGS, SGC-7901, MGC-803, MKN-45, HGC-27). (B) qPCR results in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells indicated that mRNA levels of β-catenin remained unchanged. (C) The results of western blot assays in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells indicated that protein levels of β-catenin remained unchanged (β-catenin Antibody: CST #8480S). (D) FOXC1 KO increased β-catenin levels in the cytoplasm but decreased them in the nucleus (β-catenin Antibody: CST #8480S). (E) Immunofluorescence assay in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells showed that the level of β-catenin translocation into nucleus changed with knockout and overexpression FOXC1 (β-catenin Antibody: CST #8480S). (F) FOXC1 overexpression inhibit the phospho-β-Catenin (Ser33/37/Thr41) in AGS and MKN-45 cells (phospho-β-Catenin (Ser33/37/Thr41) Antibody: CST #9561T). (G,H) co-IP assays and immunofluorescence assay indicated that FOXC1 binds to the β-catenin protein in the cytoplasm (β-catenin Antibody: CST #8084S; FOXC1 Antibody: Abcam #ab227977). (I) Western blot assays and immunoprecipitation assay were applied to confirmed the distribution of FOXC1 in the nucleus. (J) co-IP assay was applied to confirmed the distribution of phospho-β-Catenin (Ser33/37/Thr41) in the nucleus (phospho-β-Catenin (Ser33/37/Thr41) Antibody: CST #9561T). (K) co-IP assay was applied to confirmed whether FOXC1 bound to the β-catenin protein in the cytoplasm and nucleus. Immunoprecipitation assay results showed that FOXC1 bound to non-phospho-β-Catenin in the cytoplasm (β-catenin Antibody: CST #8084S; phospho-β-Catenin (Ser33/37/Thr41) Antibody: CST #9561T; Non-phospho-β-Catenin (Ser33/37/Thr41) Antibody: CST #8814T), and FOXC1 did not bound to β-Catenin in the nucleus (β-catenin Antibody: CST #8084S). *P < 0.05, **P < 0.01, ***P < 0.001.