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) Wnt3a/sFRP2 complexes interact with HSPG on the cell surface, while Afamin/Wnt3a shows no interaction. (B) Schematic illustration of the membrane-tethered HepIII system (top). HepIII-HA-GPI was stably expressed in detector cells and wild type L929 cells to enzymatically degrade cell-surface heparan sulfate. HepIII specifically cleaves the glycosidic bonds of heparin and heparan sulfate. Impact of heparan sulfate depletion on detector activation (bottom). mCherry induction levels were compared between standard detector cells and HepIII-expressing detector cells using flow cytometry. The mCherry expression was significantly reduced in HepIII-expressing cells, although it was not completely inhibited to basal levels. (C) Quantification of GFP-Wnt3a recruitment and internalization. After treating L929 cells with conditioned medium for 24 hours, confocal images were acquired for the GFP channel and differential interference contrast (DIC) (left). Scale bar: 20 µm. The total GFP intensity in each single cell was quantified after performing segmentation (right). Treatment with GFP-Wnt3a CM alone slightly increased GFP signals compared to the mock control, while the combined treatment with sFRP2 CM further enhanced these signals. HepIII expression resulted in a slight reduction of GFP recruitment specifically in the combined treatment group. Statistical significance was determined using Welch’s t-test with *** for P < 0.001, * for P < 0.05, and n.s. (non-significant) for P ≧ 0.05. (D) An mCherry-sFRP2 construct was generated by fusing mCherry to the N-terminus of sFRP2 to visualize the distribution of Wnt3a and sFRP2 at the same time. The intracellular localization of these proteins was monitored using confocal microscopy. Representative images display GFP-Wnt3a alone (left), mCherry-sFRP2 alone (center), and the combined treatment of GFP-Wnt3a and mCherry-sFRP2 (right). The colocalization of green and red signals results in yellow fluorescence, appearing as small puncta within the perinuclear region (white arrowheads). Scale bar: 20 μm. To quantitatively evaluate the interaction between GFP-Wnt3a and mCherry-sFRP2, each single cell was automatically segmented using CellPose2.0, then Pearson’s correlation coefficients were calculated. To exclude the possibility of coincidental overlap, the correlation values obtained from the original dual-channel images were compared against those derived from randomly shuffled GFP and mCherry channels.

Article Snippet: Organoid phenotypes were evaluated by supplementing the ENR medium with recombinant Afamin/Wnt3a and mouse sFRP2 (R&D Systems, #1169-FR-025) at the indicated concentrations.

Techniques: Membrane, Stable Transfection, Activation Assay, Expressing, Flow Cytometry, Single Cell, Control, Construct, Generated, Confocal Microscopy, Fluorescence, Derivative Assay

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) TOPFlash reporter assay with recombinant Afamin/Wnt3a and recombinant sFRP2. The reporter activity was evaluated across a range of recombinant Afamin/Wnt3a concentrations in the presence or absence of 400 ng/mL sFRP2. After 48-h incubation, the mCherry reporter activation was measured by flow cytometry. sFRP2 significantly enhanced reporter activity, with the most significant effect observed at an Afamin/Wnt3a concentration of 300 ng/mL. This synergistic effect is consistent with results obtained from experiments using conditioned media harvested from GFP-Wnt3a-secreting cells. (B) Experimental timeline and organoid culture workflow. When routinely-cultured intestinal organoids with IntestiCult reached passage timing, dissociated organoids were cultured with ERN medium in a 96-well plate. Various concentrations of Afamin/Wnt3a and sFRP2 were supplemented with ERN medium. (C) Representative organoids images with various cultured conditions at 4 days post seeding. Images were acquired every 24 hours by a microscope. Scale bar: 200 µm (D) Quantification of number of expanded organoids. The total number of viable organoids per well was calculated to assess the survival and proliferative impact of each treatment. The addition of sFRP2 led to a significant increase in the number of formed organoids compared to treatments with Wnt3a alone at 60, 300 ng/mL Afamin/Wnt3a concentration. Statistical differences were evaluated using Welch’s t-test with ** for P < 0.01, * for P < 0.05, and n.s. (non-significant) for P ≧ 0.05. Experiments were performed with triplicate, and data are presented as mean ± SD. (E) Quantitative assessment of budding morphogenesis. Organoid circularity was measured to evaluate the shift from spherical cystic structures to complex budding phenotypes. The addition of sFRP2 to Afamin/Wnt3a significantly increased circularity, mirroring the effect of high-dose Wnt3a and suggesting that sFRP2 amplifies Wnt signaling potency. Statistical differences were evaluated using Kruskal-Wallis and Dunn’s post-hoc test with *** for P < 0.001, and n.s. (non-significant) for P ≧ 0.05. Experiments were performed with triplicate, and data are presented as mean ± SD.

Article Snippet: Organoid phenotypes were evaluated by supplementing the ENR medium with recombinant Afamin/Wnt3a and mouse sFRP2 (R&D Systems, #1169-FR-025) at the indicated concentrations.

Techniques: Reporter Assay, Recombinant, Activity Assay, Incubation, Activation Assay, Flow Cytometry, Concentration Assay, Cell Culture, Microscopy