topflash mcherry reporter  (Addgene inc)

Journal: bioRxiv

Article Title: A synNotch-based morphogen detection system reveals sFRP2 enhances Wnt3a signaling

doi: 10.64898/2026.02.09.704138

Figure Lengend Snippet: (A) Schematic illustration of HEK293T cells expressing TOPFlash mCherry reporter. (B) TOPFlash reporter assay with recombinant sFRP2. Reporter cells were incubated with the conditioned media harvested from GFP-Wnt3a-secreting cells and supplemented with various concentrations of recombinant sFRP2. The conditioned media were applied either at full concentration (right) or diluted with an equivalent volume of DMEM (left). After 48-h incubation, the mCherry reporter activation was measured by flow cytometry. The potentiating effect of sFRP2 on Wnt signaling was pronounced under the diluted conditions (left). (C) Experimental setup for the gradient assay. GFP-Wnt3a-secreting L cells (1.6×10 4 cells) were seeded into the left chamber, while HEK293T TOPFlash reporter cells (1.6×10 4 cells) were seeded into the right chamber. After incubation overnight, the insert was removed, and the media were replaced with 1% agarose-containing media. Standard DMEM or DMEM containing recombinant sFRP2 was subsequently added over the solidified agarose gel. The confocal images were acquired at 24 h intervals. (D) Gradient patterns after 4 days of incubation. The spatial distribution of mCherry signals was extended in an sFRP2-dose-dependent manner. Red lines indicate the mean mCherry intensity derived from 5 subdivided vertical regions per image. Specifically, each 900-pixel vertical image was partitioned into 5 equal segments of 180 pixels each. The mean fluorescence intensity was calculated for each segment, and the red line represents the average of these five regional means. Shaded areas in the graph represent the ±SD. Scale bar: 200 µm.

Article Snippet: TOPFlash-mCherry reporter was optimized from TOPFlash-GFP reporter (Addgene, #35489).

Techniques: Expressing, Reporter Assay, Recombinant, Incubation, Concentration Assay, Activation Assay, Flow Cytometry, Agarose Gel Electrophoresis, Derivative Assay, Fluorescence

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