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: Cell & Bioscience

Article Title: β-catenin mediates endodermal commitment of human ES cells via distinct transactivation functions

doi: 10.1186/s13578-024-01279-5

Figure Lengend Snippet: Diverse rescue of DE differentiation by different β-catenin mutants. A Schematics of β-catenin protein and various engineered mutants. Different functional domains were indicated. Positions shown were amino acid residues. B TOPFlash reporter assays. Shown were the schematics of reporter construct (upper) and the luciferase activities reflecting TCF-dependent transactivation induced by FL β-catenin and various mutants (lower). The assays were performed using CTNNB1-/- HEK293T cells (Fig. S2). Data shown are mean ± SD. Student’s t-test was performed between individual test and untransfected control cells. ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. C DE induction processes from wt hESC (H1), CTNNB1-/- clone (KO#7), and single-cell clones rescued with FL β-catenin and different mutants in CTNNB1-/- hESCs (KO#7) background. Brightfield images were taken every day from d0 to d4 during DE induction. Scale bars = 100 μm. Close views of the bright field images in green and orange boxes were shown below (scale bars = 50 μm). Immunostaining of FOXA2 and SOX17 were performed at DE (d4). Nuclei were counterstained using Hoechst. Scale bars = 50 μm. D Percentage of scattered cells observed at d0, d1 and d2 during DE induction from the cell clones examined in C . The calculation was performed using Image J. E Ratio of FOXA2-positive cells at DE (d4) from the cell clones examined in C . The calculation was performed using Image J. F Immunostaining of α-catenin (red) and F-actin (green) in different rescue clones, at 12 h and 24 h after Dox treatment. Nuclei were counterstained using Hoechst (blue). Shown were images with merged signals (upper) and close views of selected areas (indicated by boxes) presented in individual channels. Scale bars = 50 μm. G Relative expression (fold) of EMT marker genes ( TWIST1, TWIST2, SNAI1, SNAI2 and VIM ) after Dox treatment for 24 h or at DE (d1). The qRT-PCR data in CTNNB1-/- hESC (KO#7) and various rescue clones were normalized to that in undifferentiated wt hESCs (H1) (red dashed line) and presented as mean ± SD (n = 3). For statistical analysis, all data were compared to that of undifferentiated wt hESCs (H1). ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001

Article Snippet: TOPFlash luciferase reporter (Fig. B) was modified from TOP-GFP plasmid (Addgene #35489) by replacing GFP with the Firefly luciferase gene.

Techniques: Functional Assay, Construct, Luciferase, Control, Clone Assay, Immunostaining, Expressing, Marker, Quantitative RT-PCR

Journal: JCI Insight

Article Title: Tubular β -catenin and FoxO3 interactions protect in chronic kidney disease

doi: 10.1172/jci.insight.135454

Figure Lengend Snippet: (A) PT cells were treated with varying doses of Wnt3a either in control conditions (PT medium as described in Methods) or with oxidative stress (H2O2 100 μM in serum-free DMEM/F12) for 16 hours, and Axin2 transcripts were measured by qPCR and normalized to Gapdh. (B) PT cells stably transfected with a Topflash reporter construct (see Methods) were treated with various doses of Wnt3a in either control or oxidative stress medium (H2O2 100 μM in serum-free DMEM/F12). A luminometer measured the TCF/LEF-dependent activity (Steady Glo), which was normalized to cell number by using Cell Titer assay. For both A and B, Holm-Šídák multiple-comparisons test was used. (C) Cells treated with AA for 5 days showed increased oxidative stress reflected by increased nitrotyrosine on immunoblots. PT cells were treated with AA (30 μM), Wnt3a (10 ng/mL) was added during the last 48 hours, and nuclei were isolated (see Methods) and immunoblotted for FoxO1, FoxO3, or histone H3 for loading (D), and the results from 3 separate experiments were quantified (E). (F) PT cells were treated ± Wnt3a (10 ng/mL) and oxidative stress (H2O2 100 μM) for 16 hours, and then nuclei were isolated and coimmunoprecipitation was performed. Nuclear isolates had either β-catenin pull-down or IgG control, then were immunoblotted with FoxO3, and histone H3 was used for loading control of nuclear input. The nuclear input was also immunoblotted with α-tubulin, to assess for nuclear purity, and β-catenin. Levels of FoxO3 from the coimmunoprecipitation, normalized to histone H3 (nuclear input), were quantified from 3 separate experiments (G), as were nuclear β-catenin levels (H). Student’s t test was used for statistical analyses in G and H, and ANOVA was used for multiple comparisons in E with *P < 0.05 and **P < 0.01.

Article Snippet: Wnt/β-catenin reporter or Topflash ( Tcf/Lef:H2B-GFP ) mice harboring H2B-EGFP fusion protein expression under the control of TCF/LEF1 response element ( 26 ) were obtained from The Jackson Laboratory.

Techniques: Control, Stable Transfection, Transfection, Construct, Activity Assay, Titer Assay, Western Blot, Isolation

Journal: Stem Cell Research & Therapy

Article Title: Molecular control of nitric oxide synthesis through eNOS and caveolin-1 interaction regulates osteogenic differentiation of adipose-derived stem cells by modulation of Wnt/β-catenin signaling

doi: 10.1186/s13287-016-0442-9

Figure Lengend Snippet: Lentiviral vectors and reporter constructs used in this study

Article Snippet: To further explore mechanisms by which NO promotes canonical Wnt signaling, we used a lentiviral vector expressing GFP reporter under the control of the TCF/LEF promoter (TOPFLASH; Addgene).

Techniques: Construct, Plasmid Preparation

Journal: Stem Cell Research & Therapy

Article Title: Molecular control of nitric oxide synthesis through eNOS and caveolin-1 interaction regulates osteogenic differentiation of adipose-derived stem cells by modulation of Wnt/β-catenin signaling

doi: 10.1186/s13287-016-0442-9

Figure Lengend Snippet: Nitric oxide signaling promotes activity of a β-catenin reporter. a Schematic of lentiviral transduction of a β-catenin reporter, TCF/LEF ( TOP-dGFP ), and a doxycycline ( DOX ) inducible endothelial nitric oxide synthase ( eNOS ) transduction and assessment of TCF/LEF activity during osteogenesis. b Relative mRNA transcript analysis by qPCR showing eNOS upregulates GFP mRNA expression when treated with DOX. * p < 0.05, versus TOP-dGFP + eNOS (no DOX), TOP-dGFP (osteogenic induction), TOP-dGFP (growth medium), and un-transduced eASC (growth medium)

Article Snippet: To further explore mechanisms by which NO promotes canonical Wnt signaling, we used a lentiviral vector expressing GFP reporter under the control of the TCF/LEF promoter (TOPFLASH; Addgene).

Techniques: Activity Assay, Transduction, Expressing