Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: HCT116-P and HCT116-MT cells show dysregulation of cell-cell adhesion-related proteins. (a) Western blot analysis of β-catenin, Claudin-7, and E-cadherin levels in three HCT116 cell lines (Parent, WT, and MT). (b) Quantification of band intensities for the western blot shown in. ( a ) Error bars represent the SD of mean band intensities obtained from three independent experiments. One-way ANOVA with a post-hoc test (Bonferroni) was performed to compare multiple means; * P < 0.01, ** P < 0.001, *** P < 0.0001. Statistical significance between low (L) and moderate (M) cell densities, and M and high (H) cell densities was shown. Statistical significance between L and H was shown when there was no statistical significance between L and M or M and H. Immunofluorescence microscopy analysis of β-catenin (stained in green) and E-cadherin (stained in red) in (c) HCT116-P, (d) HCT116-WT, and (e) HCT116-MT cells, under conditions of low, moderate, and high cell density. Immunofluorescence microscopy analysis of Claudin-7 (stained in red) in (f) HCT116-P, (g) HCT116-WT, and (h) HCT116-MT, under conditions of low, moderate, and high cell density. Nuclear DAPI staining is shown in blue. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: E-cadherin binded to both WT and mutant β-catenin, and knockdown of E-cadherin downregulated membranous β-catenin expression. (a) Immunoprecipitation was performed in HCT116-WT and HCT116-MT cells using a β-catenin antibody. (b) Schematic model of EGFP-conjugated β-catenin expression constructs. β-catenin-S45del vector was generated by mutagenesis using β-catenin-WT vector. (c–e) Western blot, qPCR, and immunofluorescence microscopy analysis of HCT116-P cells transfected with β-catenin-S45del vector or β-catenin-WT vector. (f) Immunoprecipitation was performed using an EGFP antibody in HCT116-P cells transfected with a control vector or β-catenin expression vectors. (g) Schematic model of EGFP-conjugated E-cadherin expression vector. (h , i) Immunofluorescence microscopy analysis and immunoprecipitation with a β-catenin antibody was performed in HCT116-MT cells transfected with E-cadherin expression vector. (j , k) Western blot and immunofluorescence microscopy analysis of HCT116-WT and HCT116-P cells transfected with siRNA against E-cadherin. Red and yellow arrows indicate loss of WT β-catenin and loss of E-cadherin, respectively. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Mutagenesis, Expressing, Immunoprecipitation, Construct, Plasmid Preparation, Generated, Western Blot, Immunofluorescence, Microscopy, Transfection

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: ZEB1 played important roles in mutant β-catenin-mediated loss of cell-cell junction molecules. (a , b) Cell density-dependent mRNA and protein expression of six EMT markers ( SNAI1 , SNAI2 , ZEB1 , TWIST1 , VIM , and CDH2 ) measured by qRT-PCR and western blot in HCT116-P, HCT116-WT, and HCT116-MT cells. Error bars represent the SD of the mean of two independent experiments. (c) Morphologies of HCT116-P and HCT116-MT cells after complete knockdown of β-catenin expression using shRNA. (d) A proliferation assay was performed using HCT116-P and HCT116-MT cells with or without β-catenin expression. 0.3 × 10 6 cells were seeded in 60-mm dishes and manually counted after 2 and 4 days. (e) Western blot analysis of E-cadherin, Claudin-7, SNAIL, SLUG, ZEB1, and TWIST1 in HCT116-P and HCT116-MT cells with or without β-catenin ablation. (f , g) Western blot and qPCR analysis of E-cadherin and Claudin-7 after knockdown of SNAIL, SLUG, ZEB1, and TWIST1 in HCT116-P and HCT116-MT cells. Error bars represent the SD of the mean of two independent experiments. All assays were carried out in duplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Mutagenesis, Expressing, Quantitative RT-PCR, Western Blot, shRNA, Proliferation Assay

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: Loss of tight junctions mediated by Claudin-7 dysregulation was sufficient for acquisition of mesenchymal-like features by HCT116 cells. (a) Western blot analysis of E-cadherin and Claudin-7 expression in HCT116-WT cells with stable shRNA-mediated knockdown of Claudin-7 (shCLDN7) or E-cadherin (shCDH1). (b) Morphological changes in HCT116-WT cells after stable knockdown of Claudin-7 or E-cadherin. Cell images were obtained using the 20x objective. (c) Immunofluorescence microscopy analysis of Claudin-7 (stained in red), β-catenin (stained in green), and E-cadherin (stained in red) in HCT116-WT cells with stable shRNA-mediated knockdown of Claudin-7 or E-cadherin knockdown. (d) Wound-healing assay performed using HCT116-WT cells with stable knockdown of Claudin-7 or E-cadherin. Gap closure was measured at 24, 48, and 72 h after the initial scratch, due to the relatively low migratory activity of HCT116-WT. (e) Quantification of relative migration distances for the wound-healing assay shown in. ( d ) Statistical significance between 0 and 24 hours, 24 and 48 hours, and 48 and 72 hours is shown. (f) Invasion assay performed using HCT116-WT cells with stable knockdown of Claudin-7 or E-cadherin. Invasive activity was measured after 48 h by crystal violet staining. (g) Quantification of the relative number of stained knockdown cells in ( f ) using ImageJ software. Statistical significance between shControl, shCLDN7, and shCDH1 is shown. Error bars in ( e , g ) represent the SD of the mean of results from three independent experiments. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Western Blot, Expressing, shRNA, Immunofluorescence, Microscopy, Staining, Wound Healing Assay, Activity Assay, Migration, Invasion Assay, Software

Journal: Scientific Reports

Article Title: β-catenin activation down-regulates cell-cell junction-related genes and induces epithelial-to-mesenchymal transition in colorectal cancers

doi: 10.1038/s41598-019-54890-9

Figure Lengend Snippet: Additional loss of adherens junctions (AJs) led to EMT progression in HCT116-P cells. (a) Western blot analysis of Claudin-7 and E-cadherin expression in HCT116-P cells with stable shRNA-mediated knockdown of Claudin-7 (shCLDN7) or E-cadherin (shCDH1). (b) Morphological changes in HCT116-P cells after stable knockdown of Claudin-7 or E-cadherin. (c) Immunofluorescence microscopy analysis of Claudin-7 (stained in red), β-catenin (stained in green), and E-cadherin (stained in red) in HCT116-P cells with stable shRNA-mediated knockdown of Claudin-7 or E-cadherin knockdown. (d) Wound-healing assay performed on HCT116-P cells with stable knockdown of Claudin-7 or E-cadherin. (e) Quantification of relative migration distances for the wound-healing assay knockdown shown in. ( d ) Statistical significance between 0 and 24 hours, and 24 and 48 hours is shown. (f) Invasion assay performed on HCT116-P cells with stable knockdown of Claudin-7 or E-cadherin. (g) Quantification of the relative number of knockdown stained cells in ( f ) by ImageJ software. Error bars in ( e , g ) represent the SD of the mean of results from three independent experiments. Statistical significance between shControl, shCLDN7, and shCDH1 is shown. All assays were carried out in triplicate.

Article Snippet: An EGFP-conjugated E-cadherin expression vector was purchased from Addgene (#Plasmid 20089, Watertown, MA, USA).

Techniques: Western Blot, Expressing, shRNA, Immunofluorescence, Microscopy, Staining, Wound Healing Assay, Migration, Invasion Assay, Software