Journal: PLoS ONE

Article Title: Henryin, an ent-kaurane Diterpenoid, Inhibits Wnt Signaling through Interference with β-Catenin/TCF4 Interaction in Colorectal Cancer Cells

doi: 10.1371/journal.pone.0068525

Figure Lengend Snippet: (A) Henryin does not affect the amount of total β-catenin and pho-β-catenin in SW480 cells. (B) Henryin does not affect the distribution of β-catenin in cytosol and nucleus fractions in SW480 cells. SW480 cells were treated with the indicated dosages of henryin for 24h. The cell extracts or fractions were prepared and analyzed by western blotting. Lamin A/C and β-actin were used as loading controls of nuclei and cytoplasm proteins, respectively. (C) Henryin antagonizes the Wnt signaling stimulated with LiCl or β-catenin. HEK293T cells were transiently transfected with ST-Luc and β-catenin or constitutively active β-catenin (S37A) plasmids, respectively, or treated with LiCl (20mM). Around 3h after transfection or treatment, henryin was added and cells were incubated for an additional 24h. Each bar is the mean ± SD from three independent experiments. (D) Henryin, but not enmenol and minheryin C, reduced the TCF4 levels associated with β-catenin in SW480 cells in a dose dependent manner. Cells were treated with the indicated dosages of henryin and its analogs enmenol and minheryin C for 12h and immune-precipitation was performed by β-catenin antibody with mouse IgG as a control, and then TCF4 was detected by western blotting. (E) Henryin directly disrupts the interaction of β-catenin with TCF4 in the in vitro binding assay. Human recombinant β-catenin (0.8µg) and TCF4 (0.5µg) were mixed, incubated with different concentrations of henryin and its analogs enmenol and minheryin C, and the mixture was subjected to co-immunoprecipitation with β-catenin antibody and western blotting analysis with TCF4 antibody, with mouse IgG used as control. (F) Quantification of the western blots shown in D and E. The software ImageJ was used to analyze the intensities of bands. Data was presented as mean ± SD from three experiments. Hen, henryin, En, enmenol, Min, minheryin C.

Article Snippet: SW480 and HCT116 cells were co-transfected with 100ng of plasmids in total, including 80ng of a well-characterized Wnt/β-catenin pathway responsive firefly luciferase reporter plasmid SuperTOPflash (ST-Luc) and 20ng of pRL-SV40 (Promega) control reporter plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Incubation, In Vitro, Binding Assay, Recombinant, Immunoprecipitation, Software

Journal: Molecular Vision

Article Title: Identification of two novel LRP5 mutations in families with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Failure of mutant LRP5 in activation of the Norrin/β-catenin pathway. SuperTopFlash (STF) cells cotransfected with LRP5-pRK5 and FZD4 constructs were treated with Norrin and assayed for luciferase reporter activity. Each assay was performed in triplicate at the same time and repeated three times. The results are an average of three measurements. Both of the novel LRP5 mutants failed to induce the luciferase reporter activity in STF cells in response to Norrin (87% reduction for p.A422T, 97% reduction for p.L540P). The additional LRP5 mutation in Patient 1 (p.Q816P) from the unaffected mother had similar luciferase reporter activity compared with the wild type, while the second mutation of Patient 2 (p.T852M), which was not detected in either parent, exhibited a 94.9% reduction. The luciferase intensities of the two combinations (p.A422T and p.Q816P, p.L540P and p.T852M) decreased significantly compared to the wild type, but no significant differences were observed compared with the mere mutation (p.A422T or p.L540P; p>0.05). The mutant p.R570Q (positive control) exhibited a 96% reduction of its wild-type activity, which was similar to the results of a previous report. Asterisks indicate significant differences between the mutant and wild type as judged by the Student two-tailed t test (p<0.05).

Article Snippet: The SuperTopFlash (STF) construct (generously provided by Dr. Amir Rattner and Dr. Jeremy Nathans of Johns Hopkins University) contains a firefly luciferase reporter driven by seven lymphoid enhancer factor/T cell factor proteins (LEF/TCF) consensus binding sites.

Techniques: Mutagenesis, Activation Assay, Construct, Luciferase, Activity Assay, Positive Control, Two Tailed Test

Journal: Oncogene

Article Title: The interactions of Bcl9/Bcl9L with β-catenin and Pygopus promote breast cancer growth, invasion, and metastasis

doi: 10.1038/s41388-021-02016-9

Figure Lengend Snippet: A Tcf and Smad-promoter luciferase reporter assays (Qiagen Cignal Lenti System) were used to monitor canonical Wnt-mediated and canonical TGFβ transcriptional outputs during TGFβ-induced EMT of Py2T cells. The graph represents luciferase activity units of Tcf and Smad reporters relative to negative controls Py2T cells 7 days after TGFβ treatment. Firefly luciferase activity measurements were normalized to Renilla luciferase activity. Data are presented as mean ± SEM. Statistical analysis was performed using the unpaired t -test. *** p < 0.001. B TOPflash (TCL/LEF-Firefly luciferase) and FOPflash (Neg control, mutated Tcf-Lef-binding site) luciferase reporter assay in Py2T cells treated with Wnt3a or TGFβ or both. Firefly luciferase activity measurements were normalized to Renilla luciferase activity. Data are presented as mean ± SEM. Statistical analysis was performed using the ordinary one-way ANOVA multiple comparison test. * p < 0.05; ** p < 0.01; *** p < 0.001. C siRNA-mediated depletion of Bcl9 and Bcl9L prevents TGFβ-induced EMT. NMuMG/E9 cells were transfected with control siRNA (siCtrl) or siRNAs against Bcl9 (siB9), Bcl9L (siB9L) or both (siB9/B9L) 2 days before starting the TGFβ treatment for further 4 days. Immunofluorescence microscopy analysis visualized the epithelial markers tight junction protein-1 (ZO-1) and E-cadherin and the mesenchymal marker Vimentin. Fluorescently labeled phalloidin visualized the actin cytoskeleton, and nuclei were counterstained with DAPI. Scale bar, 100 µm. D siRNA-mediated depletion of Bcl9 and Bcl9L partially reverses TGFβ-induced EMT. Mesenchymal Py2T cells previously treated for >20 days (Py2T-LT) were transfected with control siRNA (siCtrl) or siRNAs against Bcl9 (siB9), Bcl9L (siB9L), or both (siB9/B9L). Immunofluorescence microscopy analysis visualized the epithelial markers tight junction protein-1 (ZO-1) and the mesenchymal marker Vimentin. Nuclei were counterstained with DAPI. Scale bar, 100 µm. E siRNA-mediated depletion of Bcl9 and Bcl9L reduces migration of Py2T-LT cells. Untreated epithelial Py2T cells or mesenchymal Py2T cells previously treated for >20 days (Py2T-LT) were transfected with siCtrl, siB9, siB9L or both (siB9/B9L) and allowed to migrate for 18 h along a serum gradient in a Transwell migration assay. Migrated cells were stained with DAPI and quantified relative to siCtrl-transfected cells. Data are presented as mean ± SEM. Statistical analysis was performed using ordinary one-way ANOVA multiple comparison test. * p < 0.05; ** p < 0.01.

Article Snippet: The firefly luciferase reporter constructs superTOPflash and superFOPflash (kindly provided by Konrad Basler, UZH Zürich) were used to quantify Wnt/β-catenin-mediated transcriptional output.

Techniques: Luciferase, Activity Assay, Control, Binding Assay, Reporter Assay, Comparison, Transfection, Immunofluorescence, Microscopy, Marker, Labeling, Migration, Transwell Migration Assay, Staining

Journal: Oncogene

Article Title: The interactions of Bcl9/Bcl9L with β-catenin and Pygopus promote breast cancer growth, invasion, and metastasis

doi: 10.1038/s41388-021-02016-9

Figure Lengend Snippet: A , B Primary epithelial tumor cells with B9/B9L wt/wt, wt/fl, ΔHD1/fl, ΔHD2/fl, and β-catenin fl/fl and D164A/fl genotypes were isolated from mammary tumors of the various genotype mice. B9/B9L wt/-, ΔHD1/- and ΔHD2/- and β-catenin D164A/- cell lines were generated by infection with Cre-expressing Adenoviruses (Ad-Cre-IRES-GFP). Cells with and without Cre expressions were treated with no cytokine or Wnt3a (3 days, 100 ng/ml). Tumor-derived cell lines expressing exclusively wild-type B9/B9L (wt/wt, wt/fl or wt/-) or exclusively B9/B9L mutants (ΔHD1/- and ΔHD2/-) (A) or exclusively wild-type β-catenin (fl/fl-1 and fl/fl-2) or β-catenin-D164A (D164A/-) (B) were transfected with superTOPflash (TCL/LEF-Firefly luciferase) or superFOPflash (mutated TCF-LEF-binding site) and treatment with or without Wnt3a for 3 days and canonical Wnt signaling output was determined by bioluminescence assays. Relative transcriptional output was calculated relative to wild-type cells in the absence of Wnt3a set to 1. The right panel in B shows the lack of variation of β-catenin/Tcf-mediated transcription in the various genotype β-catenin cell lines in the absence of Wnt3a. Statistical analysis was performed using ordinary one-way ANOVA multiple comparison test. * p < 0.05; ** p < 0.01; **** p < 0.0005. C , D Cell lines derived from tumors of the various genotype MMT-PyMT transgenic mice were treated with recombinant Wnt3a (3 days; 100 ng/ml) or recombinant TGFβ (4 days; 2 ng/ml) and subjected to next-generation RNA-sequencing. Venn diagrams showing the overlap of differentially expressed genes between B9/B9L ΔHD1/-, B9/B9l ΔHD2/- and β-catenin D164A/- mutant cell lines compared to their wild-type control cell lines upon treatment with Wnt3a (C) and TGFβ (D). RNA changes with p ≤ 0.05 and a fold change ≥1.5 were considered differentially expressed. For the gene lists shared by the B9/B9L-β-catenin interaction mutants ΔHD2/- and D164/- please see Supplementary Tables and .

Article Snippet: The firefly luciferase reporter constructs superTOPflash and superFOPflash (kindly provided by Konrad Basler, UZH Zürich) were used to quantify Wnt/β-catenin-mediated transcriptional output.

Techniques: Isolation, Generated, Infection, Expressing, Derivative Assay, Transfection, Luciferase, Binding Assay, Comparison, Transgenic Assay, Recombinant, RNA Sequencing, Mutagenesis, Control