fopflash 12457 reporters  (Addgene inc)

Journal: Journal of Advanced Research

Article Title: Circular RNA circATM binds PARP1 to suppress Wnt/β-catenin signaling and induce cell cycle arrest in gastric cancer cells

doi: 10.1016/j.jare.2025.04.033

Figure Lengend Snippet: CircATM binding to PARP1 inhibits Wnt/β-catenin signaling. (A) Western blotting was used to detect β-catenin levels after PARP1 immunoprecipitation in HEK293T cells transfected with vector pLC5-ciR and pLC5-circATM plasmids. (B) Western blotting analysis of circATM binding to β-catenin in the circRNA pull-down assay. (C) Western blotting analysis of PARP1 levels in HEK293T cells transfected with vector pLC5-ciR and pLC5-circATM plasmids after β-catenin immunoprecipitation. (D) TOPflash and FOPflash reporter assays were carried out to measure the transcriptional activity of TCF/β-catenin in AGS cells. (E) Wnt/β-catenin targets were detected by RT-qPCR after circATM siRNA transfection of AGS cells. (F) Wnt/β-catenin targets were detected by western blotting after circATM siRNA transfection of AGS cells. (G) The effect of circATM overexpression on Wnt/β-catenin targets in AGS cells transfected with si_circATM. * p ＜0.05, ** p ＜0.01, *** p ＜0.001.

Article Snippet: The TOPflash (#12456) and FOPflash (#12457) reporters were purchased from Addgene (Cambridge, MA, USA).

Techniques: Binding Assay, Western Blot, Immunoprecipitation, Transfection, Plasmid Preparation, Pull Down Assay, Activity Assay, Quantitative RT-PCR, Over Expression

Journal: BMC medicine

Article Title: LASS2 enhances chemosensitivity to cisplatin by inhibiting PP2A-mediated β-catenin dephosphorylation in a subset of stem-like bladder cancer cells.

doi: 10.1186/s12916-023-03243-5

Figure Lengend Snippet: Fig. 3 LASS2 attenuates Wnt/β-catenin signaling by inhibiting β-catenin nuclear translocation. A Screening of differentially activated pathways in LASS2-overexpressing BCSCs versus control BCSCs by Cignal Finder 10-Pathway Reporter Array. B Heatmap showing the expression pattern of Wnt/β-catenin downstream genes in BCSCs transfected with the indicated plasmids. C, D Immunoblotting of the indicated cytoplasmic (C) or nuclear (D) proteins in the control or LASS2-overexpressing BCSCs treated with vehicle (PBS) or 250 ng/mL Wnt3a for 6 h. E Immunofluorescent staining images of the indicated cells treated with either Wnt3a (250 ng/mL) or vehicle (PBS) for 6 h. Scale bar, 20 μm. F, G Luciferase activity of the ABCC2-reporter plasmid in BCSCs transfected with the indicated plasmids and treated with or without Wnt3a (250 ng/mL) for 30 min. Data are presented as the means ± SD of three technical replicates. ***p < 0.001, determined by one-way ANOVA. H TOPFlash/FOPFlash reporter gene assay in control and LASS2-overexpressing BCSCs treated with Wnt3a (250 ng/mL) or vehicle (PBS) for the indicated periods. Data are presented as the means ± SD of three technical replicates. ***p < 0.001 versus vehicle, ns, not significant versus vehicle; #p < 0.05, ###p < 0.001, NS, not significant, determined by one-way ANOVA. I, J Upper panel: schematic illustrations of the primer fragments of the ABCC2 (I) and CD44 (J) promoters. Lower panel: control and LASS2-overexpressing BCSCs treated with or without Wnt3a (250 ng/ml) for 30 min were subjected to chromatin immunoprecipitation using antibodies against β-catenin and TCF4, followed by RT–PCR for ABCC2 (I) and CD44 (J) gene fragments. Data are presented as the percentage of input. Data are presented as the means ± SD of three technical replicates. ns, not significant, **p < 0.01, ***p < 0.001, determined by one-way ANOVA. K A diagram illustrating the possible mechanisms of LASS2 in regulating Wnt/β-catenin signaling

Article Snippet: For measuring Wnt/β-catenin activation, the TOPFlash reporter (#12456, Addgene), a Wnt/β-catenin pathway-responsive firefly luciferase reporter plasmid, and a mutant FOPFlash reporter (#12457, Addgene) were transfected into the indicated cells, along with Wnt3a treatment for the indicated time.

Techniques: Translocation Assay, Control, Expressing, Transfection, Western Blot, Staining, Luciferase, Activity Assay, Plasmid Preparation, Reporter Gene Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction