topflash  (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: 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: bioRxiv

Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes

doi: 10.64898/2026.02.14.705933

Figure Lengend Snippet: A) Mesenchymal cells harvested from the frontonasal mass of stage 24 (E4.5) embryos and were plated into high-density cultures. B) Other cultures were sectioned and used for microscopic analysis. C,E-H) There is a significant decrease in the proportion of cartilage from the DVL1 1519ΔT variant compared to the wtDVL1 infected cultures as measured in wholemount stained cultures. When the DVL1 1519* truncation was compared to wtDVL1 there was no significant difference in the Alcian blue stained area. The DVL1 1519* construct reduced cartilage compared to GFP controls. D,I-L) The cultures were significantly thinner in the presence of the DVL1 1519ΔT variant compared to w tDVL1 or the GFP controls. The DVL1 1519 * construct slightly reduced the thickness of the cartilage compared to GFP controls. M) The viruses containing human DVL1 constructs were expressed at similar levels in primary mesenchyme as determined by qRT-PCR with human-specific DVL1 primers. Generally the human DVL1 gene expression was elevated 10 to 15-fold by the viral transgenesis. N, O) The 1519ΔT virus significantly reduced expression of TWIST2 , MMP13 and LEF1 . P,Q) Two reporters were used in micromass cultures from frontonasal mass cells, the SuperTOPFlash reporter for canonical WNT signaling and ATF2 for JNK-PCP, non-canonical WNT signaling. The wt DVL1 plasmid significantly activated both reporters. In comparison both 1519ΔT and 1519* viruses did not activate the reporters as much as wt DVL1 . They did retain more activity than the control, parent plasmid. Statistical analysis done with one-way ANOVA followed by Dunnett’s multiple comparison test ( M ) or Tukey’s post-hoc test (C,D,N,O,P,Q). Scale bar in E-H = 2 mm, I-L = 20µm.

Article Snippet: Firefly reporter plasmids: SuperTOPFlash (STF; 0.2ug, Addgene plasmid #12456) and Activating Transcription Factor 2 (0.4ug; ATF2) ( ) along with Renilla luciferase was transfected for normalization (0.01μg).

Techniques: Variant Assay, Infection, Staining, Construct, Quantitative RT-PCR, Gene Expression, Virus, Expressing, Plasmid Preparation, Comparison, Activity Assay, Control

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