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: 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