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human recombinant protein wnt5a (R&D Systems)

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R&D Systems human recombinant protein wnt5a
Human Recombinant Protein Wnt5a, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+wnt5a/Recombinant+Human%2FMouse+Wnt-5a+Protein%2C+CF/pm41791386-192-26-30
Average 94 stars, based on 117 article reviews

human recombinant protein wnt5a - by Bioz Stars, 2026-11

94/100 stars

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Article Title: Wnt5a Promotes Axon Elongation in Coordination with the Wnt–Planar Cell Polarity Pathway
Article Snippet: Neurons were grown at 37 °C and 5% CO 2 in a serum-free neurobasal medium (Gibco, Waltham, MA, USA) supplemented with 2% B-27 (Gibco), 0.5% N-2 (Gibco), 2 mM GlutaMAX TM (Gibco), and 0.5% penicillin/streptomycin (Gibco). .. The neurons were treated with the conditioned media (CM) or recombinant Wnt5a (R&D Systems, Minneapolis, MN, USA, 645-WN-010/CF) 4 h after plating. ..

Article Title: Regionalized cell and gene signatures govern oesophageal epithelial homeostasis
Article Snippet: EN lo R lo medium contains only 10ng/ml murine Noggin (Peprotech, #250-38) and 20ng/ml recombinant R-spondin 1 (Biotechne, #3474-RS-250). .. All supplemented media are based on ENR lo medium and contain either, 250ng/ml recombinant IGF1 (Peprotech, #100-11), 250ng/ml recombinant WNT5A (R&D systems, #645-WN-010), 100ng/ml BMP4 (R&D systems, #5020-BP-010), 5ng/ml IL-17A (R&D systems, #421-ML-025/CF), or 100ng/ml recombinant NRG1 (Biotechne, #9875-NR-050). ..

Article Title: Regionalized cell and gene signatures govern esophageal epithelial homeostasis.
Article Snippet: ENloRlo medium contains only 10ng/ml murine Noggin (Peprotech, #250-38) and 20ng/ml recombinant R-spondin 1 (Biotechne, #3474-RS-250). .. All supplemented media are based on ENRlo medium and contain either, 250ng/ml recombinant IGF1 (Peprotech, #100-11), 250ng/ml recombinant WNT5A (R&D systems, #645-WN-010), 100ng/ml BMP4 (R&D systems, #5020-BP-010), 5ng/ml IL-17A (R&D systems, #421-ML-025/CF), or 100ng/ml recombinant NRG1 (Biotechne, #9875-NR-050). ..

Article Title: ROR2 homodimerization is sufficient to activate a neuronal Wnt/calcium signaling pathway.
Article Snippet: .. HEK-293 cells were transfected at 75 to 85% confluency using Lipofectamine LTX (Invitrogen) and constructs expressed for 24 to 36 h. Neurons were transfected using lipofectamine 2000 (Invitrogen), and constructs expressed for 24 to 36 h. When indicated, cells were stimulated with either 250 ng/ml of recombinant Wnt5a (R&D Systems, 645-WN-010) or 10 μm rapamycin (LC Labs, R-5000). ..

Article Title: YAP/WNT5A/FZD4 axis regulates osteogenic differentiation of human periodontal ligament cells under cyclic stretch.
Article Snippet: © 2023 John Wiley & Sons A/S.. Published by John Wiley & Sons Ltd. Xiaocen Zhang and Maolin Chang contributed equally to this work.. 1The State Key Laboratory Breeding Base of Basic Science of Stomatology (HubeiMOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China 2Orthodontic Department Division II, School & Hospital of Stomatology, Wuhan University, Wuhan, China

Article Title: Cancer-associated fibroblasts promote growth and dissemination of esophageal squamous cell carcinoma cells by secreting WNT family member 5A.
Article Snippet: Esophageal squamous cell carcinoma (ESCC) is a common and aggressive subtype of esophageal cancer.. This research investigates the functions of cancer-associated fibroblasts (CAFs) in the malignant phenotype of ESCC and probes the underpinning mechanism.. Key CAF-associated proteins in ESCC were identified using bioinformatics analyses.

Article Title: Combination treatment of chemoresistant cancers
Article Snippet: .. 300-10000 viable, single cells were plated on Ultra Low Attachment 6-well or 96-well plates (Corning Incorporated Life Sciences, Corning, NY) and cultured in MEGMTM mammary epithelial cell growth medium (Lonza, MD) with or without recombinant Wnt5a (R&D system) at 100 ng/ml for 1-3 weeks. .. Spheroids with sizes greater than 100 μm were counted using an inverted microscope (Nikon, Melville, NY).

Article Title: ROR2 homodimerization is sufficient to activate a neuronal Wnt/calcium signaling pathway
Article Snippet: .. HEK-293 cells were transfected at 75 to 85% confluency using Lipofectamine LTX (Invitrogen) and constructs expressed for 24 to 36 h. Neurons were transfected using lipofectamine 2000 (Invitrogen), and constructs expressed for 24 to 36 h. When indicated, cells were stimulated with either 250 ng/ml of recombinant Wnt5a (R&D Systems, 645-WN-010) or 10 μm rapamycin (LC Labs, R-5000). ..

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a RNA-seq was performed in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3), with a threshold of |FC| > 1.5 and adjusted P-value < 0.05. KEGG enrichment analysis of genes upregulated in Adipoq Cre ; Ctnnb1 flox/flox mice relative to control mice. b mRNA expression of Wnt5a and Fzd5 in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). c Immunoblotting for Wnt5a, pCaMKK2, CaMKK2, pAMPK, AMPK, Atgl, Sirt1, Pgc1α in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). d Wnt5a and Fzd5 expression in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). e Immunoblotting for Wnt5a, pCaMKII, CaMKII, pCaMKK2, CaMKK2, pAMPK, AMPK in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). f mRNA expression of triglyceride lipolysis genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 5–6). g mRNA expression of fatty acid β-oxidation genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 6). h Immunoblotting for Cpt1, Atgl in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). i Immunoblotting for Sirt1, Pgc1α, Pparγ, Pparɑ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). j mRNA expression of Pparγ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 7). k Schematic summary of β-catenin suppression results in activation of Wnt/Ca 2+ -AMPK axis, highlighting non-canonical pathways involved in the thermogenic response. The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were one-way ANOVA with Tukey’s correction for multiple comparisons or unpaired two-tailed t -tests.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a RNA-seq was performed in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3), with a threshold of |FC| > 1.5 and adjusted P-value < 0.05. KEGG enrichment analysis of genes upregulated in Adipoq Cre ; Ctnnb1 flox/flox mice relative to control mice. b mRNA expression of Wnt5a and Fzd5 in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). c Immunoblotting for Wnt5a, pCaMKK2, CaMKK2, pAMPK, AMPK, Atgl, Sirt1, Pgc1α in iWAT of wild-type male mice at 3, 4, 5, and 8 weeks of age ( n = 3). d Wnt5a and Fzd5 expression in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). e Immunoblotting for Wnt5a, pCaMKII, CaMKII, pCaMKK2, CaMKK2, pAMPK, AMPK in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). f mRNA expression of triglyceride lipolysis genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 5–6). g mRNA expression of fatty acid β-oxidation genes in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 6). h Immunoblotting for Cpt1, Atgl in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). i Immunoblotting for Sirt1, Pgc1α, Pparγ, Pparɑ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 3). j mRNA expression of Pparγ in iWAT of 4-week-old Adipoq Cre ; Ctnnb1 flox/flox and control mice ( n = 7). k Schematic summary of β-catenin suppression results in activation of Wnt/Ca 2+ -AMPK axis, highlighting non-canonical pathways involved in the thermogenic response. The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were one-way ANOVA with Tukey’s correction for multiple comparisons or unpaired two-tailed t -tests.

Article Snippet: For inhibition and activation experiments, once full adipogenic differentiation was confirmed, cells were treated with each of the following molecules for 4 days: MSAB (S6901,Selleck, 10 uM), BAPTA-AM (S7534, Selleck, 5 uM), Box5 (P1216, Selleck, 100 uM), Wnt5a recombinant protein (HY-P704313, MCE, 100 ng/mL) with the exception of Rosiglitazone (S2556, Selleck, 2 uM), which was added on the first day of adipogenic induction in SVFs.

Techniques: RNA Sequencing, Control, Expressing, Western Blot, Activation Assay, Two Tailed Test

a Experimental strategy to suppress β-catenin in vitro by employing adipocytes differentiated from SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), MSAB, Rosiglitazone, or MSAB plus Rosiglitazone ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to suppress β-catenin while inhibiting the Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of Rosiglitazone-differentiated adipocytes treated with vehicle (0.1% DMSO), MSAB, or MSAB plus BAPTA-AM ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots and measured maximal respiration levels across the three treatment groups (Vehicle, MSAB, MSAB and BAPTA-AM) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests or two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress β-catenin in vitro by employing adipocytes differentiated from SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), MSAB, Rosiglitazone, or MSAB plus Rosiglitazone ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to suppress β-catenin while inhibiting the Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of Rosiglitazone-differentiated adipocytes treated with vehicle (0.1% DMSO), MSAB, or MSAB plus BAPTA-AM ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots and measured maximal respiration levels across the three treatment groups (Vehicle, MSAB, MSAB and BAPTA-AM) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests or two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Techniques: In Vitro, Microscopy, Staining, Labeling, Expressing, Western Blot

a Experimental strategy to suppress Wnt5a/Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Box5, MSAB, or MSAB plus Box5 ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to supply exogenous Wnt5a recombinant protein in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Wnt5a recombinant protein, MSAB, or MSAB plus Wnt5a recombinant protein ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots, measured basal respiration and measured maximal respiration levels in cultured peri-weaning adipocytes across the six treatment groups (Vehicle, Wnt5a recombinant protein, MSAB, MSAB plus Wnt5a recombinant protein, Box5, MSAB plus Box5) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress Wnt5a/Ca²⁺ pathway in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. b Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Box5, MSAB, or MSAB plus Box5 ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). c mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). d Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( b ) ( n = 3 biological replicates). e Experimental strategy to supply exogenous Wnt5a recombinant protein in vitro by employing adipocytes differentiated from the SVFs of 4-week-old wild-type mouse iWAT. f Microscopy images of adipocytes following treatment with vehicle (0.1% DMSO), Wnt5a recombinant protein, MSAB, or MSAB plus Wnt5a recombinant protein ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 20 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 32 cells). g mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( f ) ( n = 3 biological replicates). h Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis and thermogenic protein in ( f ) ( n = 3 biological replicates). i OCR plots, measured basal respiration and measured maximal respiration levels in cultured peri-weaning adipocytes across the six treatment groups (Vehicle, Wnt5a recombinant protein, MSAB, MSAB plus Wnt5a recombinant protein, Box5, MSAB plus Box5) ( n = 3 cells). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were two-way ANOVA followed by Tukey’s multiple-comparisons test or one-way ANOVA with Tukey’s correction for multiple comparisons.

Techniques: In Vitro, Microscopy, Staining, Labeling, Expressing, Western Blot, Recombinant, Cell Culture

a Experimental strategy to suppress β-catenin using adipocytes differentiated from human subcutaneous lipoaspirates. b Microscopy images of human subcutaneous adipocytes following treatment with vehicle (0.1% DMSO) and MSAB after maturation ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 30 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 30 cells). c mRNA expression of adipogenic genes in ( b ) ( n = 3 biological replicates). d mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). e Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis, Wnt/β-catenin signaling pathway and thermogenic protein in ( b ) ( n = 3 biological replicates). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests.

Journal: bioRxiv

Article Title: Wnt/β-catenin signaling restrains developmental beige adipocyte thermogenesis and its inhibition imprints long-term energy expenditure

doi: 10.64898/2026.03.23.713637

Figure Lengend Snippet: a Experimental strategy to suppress β-catenin using adipocytes differentiated from human subcutaneous lipoaspirates. b Microscopy images of human subcutaneous adipocytes following treatment with vehicle (0.1% DMSO) and MSAB after maturation ( n = 3 biological replicates). Scale bar, 200 μm. Nile Red and DAPI were used to stain lipid droplets and nuclei, respectively ( n = 3 biological replicates). Scale bar, 30 μm. The right panel displays the statistical analysis of Nile Red-labeled lipid droplets number per cell ( n = 30 cells). c mRNA expression of adipogenic genes in ( b ) ( n = 3 biological replicates). d mRNA expression of thermogenic genes and downstream genes of Wnt/β-catenin signaling pathway in ( b ) ( n = 3 biological replicates). e Immunoblotting for Wnt5a/Ca²⁺-AMPK-PPAR axis, Wnt/β-catenin signaling pathway and thermogenic protein in ( b ) ( n = 3 biological replicates). The levels of mRNA expression are normalized to that of 36B4 . Data are the mean ± s.e.m. Statistical analyses used were unpaired two-sided Student’s t -tests.

Techniques: Microscopy, Staining, Labeling, Expressing, Western Blot

( A ) UMAP of epithelial subtypes in control and NEC mice. ( B ) Representative immunofluorescence images for Lyz (red), Epacm (green) and DAPI (blue) on paraffin-embedded distal ileum from indicated group in experiment 3. White arrow indicates the Lyz stained PCs, Scale bar: 10 μm. ( C , D ) The number of Lyz + PCs in B ( C ) and mRNA levels of Lyz ( D ) were compared among different groups. ( E ) Violin plots showed the expression of Wnt/PCP pathway genes in mouse PCs subsets revealed by scRNA-seq analysis. ( F ) The mRNA level of Damm1 was compared in IECs isolated from different groups. ( G ) Organoids derived from mice were cultured in medium with DMSO, FEX (5 μm), or FEX (10 μm) from day 1. The organoids were collected and stained for Lyz on day 7. Arrowheads point at PCs (Lyz + , in pink), scale bars: 50 μm. ( H ) qRT-PCR analysis of Daam1 in organoids cultured with DMSO, FEX (5 μm), or FEX (10 μm). ( I ) Representative organoid formation images on day 7 in different groups supplemented with ENR, Wnt5a-containing ENR, and Wnt5a-containing ENR + FEX. Representative marker genes for PCs (Lyz, green) cells are highlighted by fluorescent reporters. Arrowheads point at PCs (Lyz + , in green), scale bars: 50 μm. All statistical data are shown as the means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: NPJ Biofilms and Microbiomes

Article Title: Disruption of bile acid homeostasis potentiates Paneth cell ablation by activating the intestinal Farnesoid X receptor in necrotizing enterocolitis

doi: 10.1038/s41522-025-00904-6

Figure Lengend Snippet: ( A ) UMAP of epithelial subtypes in control and NEC mice. ( B ) Representative immunofluorescence images for Lyz (red), Epacm (green) and DAPI (blue) on paraffin-embedded distal ileum from indicated group in experiment 3. White arrow indicates the Lyz stained PCs, Scale bar: 10 μm. ( C , D ) The number of Lyz + PCs in B ( C ) and mRNA levels of Lyz ( D ) were compared among different groups. ( E ) Violin plots showed the expression of Wnt/PCP pathway genes in mouse PCs subsets revealed by scRNA-seq analysis. ( F ) The mRNA level of Damm1 was compared in IECs isolated from different groups. ( G ) Organoids derived from mice were cultured in medium with DMSO, FEX (5 μm), or FEX (10 μm) from day 1. The organoids were collected and stained for Lyz on day 7. Arrowheads point at PCs (Lyz + , in pink), scale bars: 50 μm. ( H ) qRT-PCR analysis of Daam1 in organoids cultured with DMSO, FEX (5 μm), or FEX (10 μm). ( I ) Representative organoid formation images on day 7 in different groups supplemented with ENR, Wnt5a-containing ENR, and Wnt5a-containing ENR + FEX. Representative marker genes for PCs (Lyz, green) cells are highlighted by fluorescent reporters. Arrowheads point at PCs (Lyz + , in green), scale bars: 50 μm. All statistical data are shown as the means ± SEMs, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For experiments involving Wnt5a (645-WN-010/CF, R&D Systems) ligands, the media was made by supplementing ENR media with 100 ng/ml Wnt5a.

Techniques: Control, Immunofluorescence, Staining, Expressing, Isolation, Derivative Assay, Cell Culture, Quantitative RT-PCR, Marker

a General schematic of the TetON inducible gene expression system used in this study. In the absence of doxycycline, the reverse tetracycline-controlled transactivator (rtTA) is inactive. Upon doxycycline addition, rtTA binds to the tetO promoter and activates transcription of the gene of interest, leading to protein expression. b Doxycycline-induced overexpression of HA-tagged PRICKLE3 in HEK T-REx 293 PRICKLE3 TetON cells and of PRICKLE1 in the corresponding inducible cell line. c Induction of HA-PRICKLE3 expression by doxycycline in HEK T-REx 293 PRICKLE3 TetON cells. HEK T-REx 293 wildtype (WT) cells served as a control for doxycycline effects. Arrowheads indicate phosphorylation-dependent shifts in the electrophoretic mobility of VANGL proteins. α-TUBULIN was used as a loading control. Representative result from n = 5. d , e Densitometric quantification of Western blot signals. Values were normalized to untreated cells. Statistical analysis was performed using an unpaired t -test; corresponding p -values are shown ( n = 5). f Effect of recombinant WNT stimulation on VANGL phosphorylation. HEK T-REx 293 PRICKLE3 TetON cells were pre-treated overnight with the porcupine inhibitor LGK-974 to block endogenous WNT ligand secretion and subsequently stimulated with 100 ng/ml human recombinant WNT5A or WNT3A for 3 h. Arrowheads indicate phosphorylation-dependent mobility shifts of ROR. α-TUBULIN served as a loading control. Representative result from n = 4. g , h Densitometric quantification of Western blot signals from panel f. Data were normalized to untreated controls. Statistical analysis was performed using an unpaired t -test; corresponding p -values are reported ( n = 4).

Journal: Communications Biology

Article Title: PRICKLE3 protects VANGL proteins from CK1-mediated phosphorylation and RNF43-mediated degradation

doi: 10.1038/s42003-025-09422-9

Figure Lengend Snippet: a General schematic of the TetON inducible gene expression system used in this study. In the absence of doxycycline, the reverse tetracycline-controlled transactivator (rtTA) is inactive. Upon doxycycline addition, rtTA binds to the tetO promoter and activates transcription of the gene of interest, leading to protein expression. b Doxycycline-induced overexpression of HA-tagged PRICKLE3 in HEK T-REx 293 PRICKLE3 TetON cells and of PRICKLE1 in the corresponding inducible cell line. c Induction of HA-PRICKLE3 expression by doxycycline in HEK T-REx 293 PRICKLE3 TetON cells. HEK T-REx 293 wildtype (WT) cells served as a control for doxycycline effects. Arrowheads indicate phosphorylation-dependent shifts in the electrophoretic mobility of VANGL proteins. α-TUBULIN was used as a loading control. Representative result from n = 5. d , e Densitometric quantification of Western blot signals. Values were normalized to untreated cells. Statistical analysis was performed using an unpaired t -test; corresponding p -values are shown ( n = 5). f Effect of recombinant WNT stimulation on VANGL phosphorylation. HEK T-REx 293 PRICKLE3 TetON cells were pre-treated overnight with the porcupine inhibitor LGK-974 to block endogenous WNT ligand secretion and subsequently stimulated with 100 ng/ml human recombinant WNT5A or WNT3A for 3 h. Arrowheads indicate phosphorylation-dependent mobility shifts of ROR. α-TUBULIN served as a loading control. Representative result from n = 4. g , h Densitometric quantification of Western blot signals from panel f. Data were normalized to untreated controls. Statistical analysis was performed using an unpaired t -test; corresponding p -values are reported ( n = 4).

Article Snippet: Following this, non-canonical WNT signaling was activated by incubating the cells with recombinant human WNT5A (645-WN, R&D Systems) at a concentration of 100 ng/mL for 3 h. Similarly, canonical WNT signaling was activated by incubating the cells with 100 ng/mL of recombinant human WNT3A (5036-WN, R&D Systems) for 3 h. To assess protein degradation kinetics, cells were pretreated with doxycycline overnight.

Techniques: Gene Expression, Expressing, Over Expression, Control, Phospho-proteomics, Western Blot, Recombinant, Blocking Assay

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