fopflash 12457 reporters  (Addgene inc)

Journal: Chemmedchem

Article Title: 4‐(5‐Chloro‐3‐(3,4,5‐trimethoxybenzoyl)‐1 H ‐indol‐1‐yl)benzenesulfonamide: A Novel Polypharmacology Agent to Target Carbonic Anhydrase IX and XII With Improved Selectivity, Wnt/β‐Catenin Signaling Pathway, and P‐Glycoprotein

doi: 10.1002/cmdc.202500996

Figure Lengend Snippet: Compound 15 inhibits Wnt/β‐catenin activity luciferase assay in HEK‐293T cells transfected with M50 Super 8x TOPFlash (TOP) or its mutated version M51 Super 8x FOPFlash (FOP) used as negative control. Cells were induced with LiCl (50 mM) and treated with the indicated concentrations of 15 for 24 h. Data are represented as the mean ± SD of three independent experiments, each performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.005, ns not significant as determined by analysis of variance (ANOVA).

Article Snippet: HEK293T cells were transfected with M50 Super 8x TOPFlash (Addgene #12456) or the FOP control plasmid (M51 Super 8x FOPFlash Addgene #12457) in combination with TK Renilla (Promega #E2241) using DreamFect Gold (OZ Biosciences #DG80500) according to the manufacturer's instructions.

Techniques: Activity Assay, Luciferase, Transfection, Negative Control

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: Scientific Reports

Article Title: Smurf1 promotes gastric cancer progression by regulating Axin2-dependent Wnt signaling pathway

doi: 10.1038/s41598-025-23707-3

Figure Lengend Snippet: Smurf1 promoted Wnt/β-catenin signaling activation. ( A ) Parental and Smurf1-overexpressing AGS cells were transfected with TOPflash and FOPflash reporters and treated with Wnt3a, respectively. Wnt/β-catenin signaling activation was assessed using the TOPflash/FOPflash dual luciferase reporter system. Relative luciferase units (RLU) were calculated to assess β-catenin-triggered transcription. After Smurf1 overexpression in AGS cells, cytoplasmic ( B and D ) and nuclear ( C and D ) β-catenin protein levels were measured using the western blot assay. β-actin and Lamin B1 served as cytoplasmic and nuclear markers, respectively. ( E ) After Smurf1 overexpression in AGS cells, β-catenin nuclear translocation was assessed using IF analysis. Red fluorescence indicated β-catenin, and blue fluorescence indicated the nucleus. The results were shown as the median (1st quartile and 3rd quartile). * p < 0.05, ** p < 0.01.

Article Snippet: After Smurf1 overexpression, AGS cells were co-transfected with TOPflash construct (80 ng, Merck) or FOPflash construct (Merck) and pRL-TK plasmid (8 ng, Promega, WI, USA) using PEI max transfection reagent.

Techniques: Activation Assay, Transfection, Luciferase, Over Expression, Western Blot, Translocation Assay, Fluorescence