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m50 super 8x topflash  (Addgene inc)


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    Structured Review

    Addgene inc m50 super 8x topflash
    Compound 15 inhibits Wnt/β‐catenin activity luciferase assay in HEK‐293T cells transfected with <t>M50</t> 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).
    M50 Super 8x Topflash, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 579 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/topflash/M50+Super+8x+TOPFlash+(Plasmid+%2312456)/pmc12935169-352-5-9
    Average 96 stars, based on 579 article reviews
    m50 super 8x topflash - by Bioz Stars, 2026-11
    96/100 stars

    Images

    1) Product Images from "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"

    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

    Journal: Chemmedchem

    doi: 10.1002/cmdc.202500996

    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).
    Figure Legend 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).

    Techniques Used: Activity Assay, Luciferase, Transfection, Negative Control

    Related Articles

    Transfection:

    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
    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. .. After 24 h, cells were incubated with starvation media (Opti‐MEM reduced serum medium supplemented with 0.5% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids) for 8 h. After starvation, Wnt/β‐catenin signaling was activated by treating cells with 50 mM LiCl in starvation media for 24 h. The following day, cells were treated with 15 or vehicle (DMSO) for 24 h. At the end of the experiment, cells were lysed using Passive Lysis Buffer (Biotium #99912).

    Article Title: NR3C1 Modulates Wnt Signalling to Influence the Invasiveness and Immune Features of Nonfunctioning Invasive Pituitary Adenomas.
    Article Snippet: .. Cells were co- transfected using Lipofectamine 3000 (Thermo Fisher, L3000015) with either TOPflash (Addgene, #12456) or FOPflash (Addgene, #12457) (500 ng each) and the Renilla internal control vector (Promega, E2241, pRL- TK, 50 ng), along with sh- NC or sh- NR3C1 as required. .. Cells were lysed 48 h post- transfection, and luciferase activity was measured using the Promega Dual- Luciferase Reporter Assay System (Promega, E1910).

    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes
    Article Snippet: Micromass cultures were allowed to attach for 45 mins after plating and transfection reagents were added to the culture spot 30 mins prior to flooding the culture plate with micromass media. .. 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). .. 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).

    Control:

    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
    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. .. After 24 h, cells were incubated with starvation media (Opti‐MEM reduced serum medium supplemented with 0.5% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids) for 8 h. After starvation, Wnt/β‐catenin signaling was activated by treating cells with 50 mM LiCl in starvation media for 24 h. The following day, cells were treated with 15 or vehicle (DMSO) for 24 h. At the end of the experiment, cells were lysed using Passive Lysis Buffer (Biotium #99912).

    Article Title: NR3C1 Modulates Wnt Signalling to Influence the Invasiveness and Immune Features of Nonfunctioning Invasive Pituitary Adenomas.
    Article Snippet: .. Cells were co- transfected using Lipofectamine 3000 (Thermo Fisher, L3000015) with either TOPflash (Addgene, #12456) or FOPflash (Addgene, #12457) (500 ng each) and the Renilla internal control vector (Promega, E2241, pRL- TK, 50 ng), along with sh- NC or sh- NR3C1 as required. .. Cells were lysed 48 h post- transfection, and luciferase activity was measured using the Promega Dual- Luciferase Reporter Assay System (Promega, E1910).

    Plasmid Preparation:

    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
    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. .. After 24 h, cells were incubated with starvation media (Opti‐MEM reduced serum medium supplemented with 0.5% FBS, 1% penicillin/streptomycin, 1% sodium pyruvate, and 1% nonessential amino acids) for 8 h. After starvation, Wnt/β‐catenin signaling was activated by treating cells with 50 mM LiCl in starvation media for 24 h. The following day, cells were treated with 15 or vehicle (DMSO) for 24 h. At the end of the experiment, cells were lysed using Passive Lysis Buffer (Biotium #99912).

    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes
    Article Snippet: Micromass cultures were allowed to attach for 45 mins after plating and transfection reagents were added to the culture spot 30 mins prior to flooding the culture plate with micromass media. .. 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). .. 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).

    other:

    Article Title: D-glucose uptake inhibits bovine alphaherpesvirus 1 post-binding cell process entry via inhibition of PLC-γ1 signaling in a glucose transporter 1-independent manner
    Article Snippet: The plasmid S33Y, which expresses a constitutively active Flag-tagged β-catenin protein, was a gift from Bert Vogelstein (Addgene plasmid number 16519) ( ).

    Article Title: Architecture of the Norrin signaling complex and structure-guided surrogate design.
    Article Snippet: Norrin binds Frizzled 4 (FZD4), tetraspanin 12 (TSPAN12) and low-density lipoprotein receptor-related protein 5 (LRP5) to regulate angiogenesis and the integrity of the blood–brain barrier and inner blood–retina barrier (BRB).. Aberrant Norrin signal causes retinopathies, BRB defects and deafness.. Therapeutic mimetics of Norrin are currently in clinical trials for eye diseases.

    Recombinant:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    Lysis:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    Membrane:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    Sensitivity:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    Staining:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    BIA-KA:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    Real-time Polymerase Chain Reaction:

    Article Title: Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.
    Article Snippet: Antibodies Ms anti-β-actin (1:5000) Cell signal Cat. 3700S Ms anti-R-Spondin1 (1:2000) R&D systems Cat. MAB4658 Ms anti-His-tag (1:1000) Cell signal Cat. 2366 Rb anti-Non-phospho (Active) β-Catenin (Ser45) (1:1000) Cell signal Cat. 19807S Rb anti-β-Catenin (1:1000) Cell signal Cat. 8480S Rb anti-TSG101 (1:2000) Abcam Cat. ab125011 Rb Anti-CD63 (1:2000) Abcam Cat. ab134045 Rb Anti-CD9 (1:1000) Abcam Cat. ab236630 Rb anti-Calnexin (1:1000) Abcam Cat. ab133615 Rb anti-HSP70 (1:1000) Abcam Cat. ab181606 Rb anti-Ki67 (1:1000) Abcam Cat. ab15580 Rb anti-Alix (1:200) Abcam Cat. ab275377 Rb anti-p21 (1:200) Abcam Cat. ab188224 Rb anti-Olfm4 (1:500) Cell signal Cat. 39141S Rb anti-CD45 (1:500) Abcam Cat. ab10558 Ms anti-IFNγ (1:500) Santa Cruz Cat. Sc-8423 Ms anti-ZO-1 (1:500) Invitrogen Cat. XL361204 Rb anti-Occludin (1:500) Abcam Cat. ab216327 Rb anti-Muc2 (1:500) Abcam Cat. ab272692 Alexa Fluor 647-conjugated anti-CD63 antibody (1:200) Abcam Cat. ab233056 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:200) Invitrogen Cat. A-11008 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200) Invitrogen Cat. A-11012 HRP-conjugated Goat anti-Rabbit antibody (1:1000) Beyotime Cat. A0208 HRP-conjugated Goat anti-Mouse antibody (1:1000) Beyotime Cat. A0216 Chemicals, peptides, and recombinant proteins Dulbecco’s modified Eagle’s medium (DMEM) Cytiva Cat. SH20243.01 Fetal bovine serum Corning Cat. 35-179-CV Penicillin/streptomycin Gibco Cat. 15140-122 Puromycin Gibco Cat. A1113803 (Continued on next page) STAR Protocols 7, 104891, December 18, 2026 7 .. TrypLE Select Gibco Cat. A1217701 Serum-free medium for HEK293 Bioengine Cat. TP0103001 Opti-MEM Gibco Cat. 11058021 psPAX2 Addgene Cat. 12260 VSVG Addgene Cat. 8454 M50 Super 83TOPFlash Addgene Cat. 12456 Lipo8000 Beyotime Cat. C0533 Polybrene MedChem Cat. HY-K2032 Goat Anti-Mouse Ig (10nm Gold) Abcam Cat. ab39619 Heparinase I and III Blend Sigma Cat. H3917 Growth factor-reduced Matrigel Corning Cat. 356231 Advanced DMEM/F12 Gibco Cat. 12634028 EGF recombinant protein Novoprotein Cat. C029 Noggin recombinant protein Novoprotein Cat. CB89 RSPO1 recombinant protein Novoprotein Cat. CX83 N2 Gibco Cat. 17502048 B27 Gibco Cat. 17504044 GlutaMAX Gibco Cat. 35050061 N-acetylcysteine Sigma Cat. A0737 PKH26 Sigma Cat. MKCV7372 DAPI Sigma Cat. D9542 FITC-Phalloidin Beyotime Cat. C2221S Trizol Invitrogen Cat. 15596018CN DiR Meilun Bio Cat. MB12482 RIPA lysis buffer Beyotime Cat. P0013B Protease inhibitors Beyotime Cat. P1005 53loading buffer Beyotime Cat. P0015L Uranyl acetate MedChem Cat. HY-184835 Glutaraldehyde MedChem Cat. HY-Y1017 BSA Sangon Cat. A602448 203TBS buffer Sangon Cat. B548105 Tween-20 Sangon Cat. A600560 0.22-μm Filter Unit Millipore Cat. SLGPR33RB 0.45-μm Filter Unit Millipore Cat. SLHVR33RS 70-μm cell strainer BD Biosciences Cat. 352350 0.45-μm PVDF membrane Millipore Cat. IPVH00010 96-well plate Costar Cat. 3799 24-well plate Falcon Cat. 353047 6-well plate Falcon Cat. 353046 10-cm dish Corning Cat. 430167 35-mm dish Corning Cat. 430165 250-mL Erlenmeyer Flasks Biofl Cat. TAB102250 Critical commercial assays Dual-Luciferase Report Assay System Promega Cat. E1501 RSPO1 enzyme-linked immunosorbent assay kit R&D Systems Cat. DY4645-05 High Sensitivity ECL Substrate Kit Abcam Cat. ab133406 β-Galactosidase staining Servicebio Cat. G1073 BCA assay kit Beyotime Cat. P0010 HiScript II cDNA kit Vazyme Cat. R233-01 SYBR qPCR Master Mix Vazyme Cat. Q711-03 mRNA Library Fast kit Epibiotek Cat. R1810 Experimental models: Cell lines HEK293 cells Procell system CL-0001 HEK293T cells Procell system CL-0005 (Continued on next page) 8 STAR Protocols 7, 104891, December 18, 2026 ..

    Luciferase:

    Article Title: The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes
    Article Snippet: Micromass cultures were allowed to attach for 45 mins after plating and transfection reagents were added to the culture spot 30 mins prior to flooding the culture plate with micromass media. .. 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). .. 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).



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    Topflash Mcherry Reporter, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/topflash/TOP-GFP+(Plasmid+%2335489)/bio_rxiv__64898__2026__02__09__704138-130-0-7
    Average 93 stars, based on 1 article reviews
    topflash mcherry reporter - by Bioz Stars, 2026-11
    93/100 stars
    		  Buy from Supplier
    topflash gfp reporter  (Addgene inc)
    93
    Addgene inc topflash gfp reporter
    (A) Schematic illustration of HEK293T cells expressing <t>TOPFlash</t> <t>mCherry</t> 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.
    Topflash Gfp Reporter, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/topflash/TOP-GFP+(Plasmid+%2335489)/bio_rxiv__64898__2026__02__09__704138-130-5-7
    Average 93 stars, based on 1 article reviews
    topflash gfp reporter - by Bioz Stars, 2026-11
    93/100 stars
    		  Buy from Supplier

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    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).

    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

    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).

    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

    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.

    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

    (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.

    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