Journal: Poultry Science

Article Title: Frizzled-4 promotes bone formation in chickens via activation of the canonical Wnt signaling pathway

doi: 10.1016/j.psj.2026.106589

Figure Lengend Snippet: Isolation, Culture, and In Vitro Osteogenic Differentiation of Chicken BMSCs. (A) Morphological characteristics of BMSCs, (1) - (5) represent features at 0, 1, 5, 7, and 9 days post-culture, respectively. (Scale bar = 50 μm) (B) Immunofluorescence; CD29: Also known as beta-1 integrin, a positive surface marker for mesenchymal stem cells. CD45: Also known as Leukocyte Common Antigen (LCA), a hematopoietic cell marker. (C) ALP staining and ARS staining images after induced differentiation (Scale bar = 100 μm). (D-E) Relative expression of osteogenic differentiation marker gene ALP and target gene FZD4 at 0 and 3 days post-induced differentiation. n = 3. (F) Western blot analysis of FZD4 protein levels at 36 hours post-induced differentiation. (G) Gray-scale analysis of FZD4 protein bands. ImageJ software was used for band analysis. * P < 0.05, ** P < 0.01. n = 3.

Article Snippet: The primary antibodies used were FZD4 rabbit polyclonal antibody (1:2000, bs-13217R, Bioss), Glycogen synthase kinase 3 beta ( GSK-3β ) rabbit polyclonal antibody (1:2000, 51065-1-AP, Proteintech), β-catenin rabbit polyclonal antibody (1:5000, 51067-2-AP, Proteintech) and GAPDH mouse monoclonal antibody (1:10000, 60004-1-Ig, Proteintech).

Techniques: Isolation, In Vitro, Immunofluorescence, Marker, Staining, Expressing, Western Blot, Software

Journal: Poultry Science

Article Title: Frizzled-4 promotes bone formation in chickens via activation of the canonical Wnt signaling pathway

doi: 10.1016/j.psj.2026.106589

Figure Lengend Snippet: Effects of FZD4 overexpression and knockdown on the osteogenic differentiation of chicken BMSCs. (A) FZD4 expression in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 3. (B-C) Relative expression of osteogenic marker genes Col1A1, Runx2, ALP , and OCN in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (D-E) ALP staining and grayscale analysis at 7 days post-induction. n = 4. (F-G) ARS staining and grayscale analysis at 14 days post-induction. Grayscale analysis was performed using ImageJ software. n = 4. (H) FZD4 expression in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 3. (I-J) Relative expression of osteogenic marker genes Col1A1, Runx2, ALP , and OCN in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (K-L) ALP staining and grayscale analysis at 7 days post-induction (Scale bar = 100 μm). n = 4. (M-N) ARS staining and grayscale analysis at 14 days post-induction (Scale bar = 100 μm). n = 4.

Article Snippet: The primary antibodies used were FZD4 rabbit polyclonal antibody (1:2000, bs-13217R, Bioss), Glycogen synthase kinase 3 beta ( GSK-3β ) rabbit polyclonal antibody (1:2000, 51065-1-AP, Proteintech), β-catenin rabbit polyclonal antibody (1:5000, 51067-2-AP, Proteintech) and GAPDH mouse monoclonal antibody (1:10000, 60004-1-Ig, Proteintech).

Techniques: Over Expression, Knockdown, Expressing, Marker, Staining, Software

Journal: Poultry Science

Article Title: Frizzled-4 promotes bone formation in chickens via activation of the canonical Wnt signaling pathway

doi: 10.1016/j.psj.2026.106589

Figure Lengend Snippet: Effects of FZD4 overexpression and knockdown on the expression of key factors in the canonical Wnt signaling pathway. (A-B) Relative expression of signaling pathway key factors C-myc, CyclinD1, β-catenin , and GSK-3β in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (C) Western blot analysis of FZD4, GSK-3β, and β-catenin protein levels at 36 hours post-differentiation induction. (D-E) Band intensity analysis of FZD4, GSK-3β, and β-catenin proteins. n = 3. (F-G) Relative expression of key signaling pathway factors C-myc, CyclinD1, β-catenin , and GSK-3β in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (H) Western blot analysis of FZD4, GSK-3β, and β-catenin protein levels at 36 hours post-differentiation induction. (I-J) Grayscale analysis of FZD4, GSK-3β, and β-catenin protein bands. Band intensity was analyzed using ImageJ software. * P < 0.05, ** P < 0.01. n = 3.

Article Snippet: The primary antibodies used were FZD4 rabbit polyclonal antibody (1:2000, bs-13217R, Bioss), Glycogen synthase kinase 3 beta ( GSK-3β ) rabbit polyclonal antibody (1:2000, 51065-1-AP, Proteintech), β-catenin rabbit polyclonal antibody (1:5000, 51067-2-AP, Proteintech) and GAPDH mouse monoclonal antibody (1:10000, 60004-1-Ig, Proteintech).

Techniques: Over Expression, Knockdown, Expressing, Western Blot, Software

Journal: Poultry Science

Article Title: Frizzled-4 promotes bone formation in chickens via activation of the canonical Wnt signaling pathway

doi: 10.1016/j.psj.2026.106589

Figure Lengend Snippet: Effects of overexpressing FZD4 while inhibiting the canonical Wnt signaling pathway on the osteogenic differentiation of chicken BMSCs. (A-B) Relative expression of osteogenic marker genes Col1A1, Runx2, ALP , and OCN in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (C-D) Relative expression of key signaling pathway factors C-myc, CyclinD1, β-catenin , and GSK-3β in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (E-G) Western blot analysis of β-catenin and GSK-3β protein levels at 36 h post-differentiation induction. Protein band grayscale analysis was performed using ImageJ software. n = 3. (H-I) ALP staining images (Scale bar = 100 μm) and grayscale analysis at 7 days post-induction. n = 4. (J-K) ARS staining (Scale bar = 100 μm) and grayscale analysis at 14 days post-induction. * P < 0.05, ** P < 0.01. n = 4.

Article Snippet: The primary antibodies used were FZD4 rabbit polyclonal antibody (1:2000, bs-13217R, Bioss), Glycogen synthase kinase 3 beta ( GSK-3β ) rabbit polyclonal antibody (1:2000, 51065-1-AP, Proteintech), β-catenin rabbit polyclonal antibody (1:5000, 51067-2-AP, Proteintech) and GAPDH mouse monoclonal antibody (1:10000, 60004-1-Ig, Proteintech).

Techniques: Expressing, Marker, Western Blot, Software, Staining

Journal: Poultry Science

Article Title: Frizzled-4 promotes bone formation in chickens via activation of the canonical Wnt signaling pathway

doi: 10.1016/j.psj.2026.106589

Figure Lengend Snippet: Effects of knockdown FZD4 while inhibiting the canonical Wnt signaling pathway on the osteogenic differentiation of chicken BMSCs. (A-B) Relative expression of osteogenic marker genes Col1A1, Runx2, ALP , and OCN in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (C-D) Relative expression of key signaling pathway factors C-myc, CyclinD1, β-catenin , and GSK-3β in chicken BMSCs at 0 and 7 days post-osteogenic differentiation. n = 6. (E-G) Western blot analysis of β-catenin and GSK-3β protein levels at 36 h post-differentiation induction. Protein band grayscale analysis was performed using ImageJ software. n = 3. (H-I) ALP staining images (Scale bar = 100 μm) and grayscale analysis at 7 days post-induction. n = 4. (J-K) ARS staining (Scale bar = 100 μm) and grayscale analysis at 14 days post-induction. * P < 0.05, ** P < 0.01. n = 4.

Article Snippet: The primary antibodies used were FZD4 rabbit polyclonal antibody (1:2000, bs-13217R, Bioss), Glycogen synthase kinase 3 beta ( GSK-3β ) rabbit polyclonal antibody (1:2000, 51065-1-AP, Proteintech), β-catenin rabbit polyclonal antibody (1:5000, 51067-2-AP, Proteintech) and GAPDH mouse monoclonal antibody (1:10000, 60004-1-Ig, Proteintech).

Techniques: Knockdown, Expressing, Marker, Western Blot, Software, Staining

Journal: Biochemistry and Biophysics Reports

Article Title: Comparative analysis of activation of macrophages/microglia in diabetic retinopathy and Familial Exudative Vitreoretinopathy

doi: 10.1016/j.bbrep.2025.102396

Figure Lengend Snippet: FZD4 knockout mice develop FEVR features. FZD4 knockout mice (FZD4KO) was used as an FEVR model. (A) Western blot for FZD4 in the retinal tissue of FZD4 knockout mice, compared to wildtype controls (WT). (B–C) Electroretinogram (ERG) assessments at 16 weeks, shown by representative chars (B) and by quantification (C). Blue circle: b-wave; green circle: a-wave. (D) Retinal vascular density by CD31 + area quantification. N = 5. ∗p < 0.05. NS: no significant.

Article Snippet: Knockout of FZD4 in these mice were validated by Western blot for FZD4 in the retinal tissue, using monoclonal antibody against mouse FZD4 (MAB195–050, R&D Systems).

Techniques: Knock-Out, Western Blot

Journal: bioRxiv

Article Title: Cell-autonomous Wnt activity promotes transient re-programming and cell cycle re-entry of coronary artery endothelial cells

doi: 10.64898/2026.02.23.707374

Figure Lengend Snippet: ( A ) Violin plot showing levels of mRNA expression of Frizzled (Fzd) receptors in neonatal hearts. ( B ) Cryosection from Cx40CreER; Rosa26 TdTomato heart showing a cross-section of a coronary artery, immunostained for Fzd4 (green). TdTomato + lineage traced arterial endothelial cells shown in red, nuclei labelled with DAPI in blue. ( C ) Experimental design to assess the role of arterial Fzd4 in artery reassembly. ( D - F ) Confocal images of neonatal Cx40CreER; Rosa26 TdTomato hearts labelled and traced for pre-existing artery endothelial cells in black. D shows whole heart. ( E , F ) Images of watershed from control hearts with ( E ) moderate (inset from D ) and ( F ) severe MI. ( G ) Quantification of TdTomato + collaterals per heart. p<0.0001. ( H - J ) Confocal images of neonatal Fzd4 L/L ; Cx40CreER; Rosa26 TdTomato hearts, labelled and traced for pre-existing artery endothelial cells (shown in black). H shows whole heart. ( I , J ) Images of watershed from control hearts with ( I ) moderate (inset from H ) and ( J ) severe MI. Arrowheads and brackets point to single and clusters of saECs, respectively. All 5 knockout hearts undergoing moderate MI demonstrate a phenotype as shown in I . 4 out of 6 knockout hearts undergoing severe MI demonstrate a phenotype as shown in J. ( K ) Confocal image of Fzd4 depleted single artery cells, post-MI show non-EC morphology. ( L , M ) Confocal images of watershed regions from ( L ) control and ( M ) arterial knockouts for Fzd4, showing EdU + proliferating cells in green and TdTomato + lineage traced artery endothelial cells in red. Arrowheads point to EdU + TdTomato + proliferating single artery cells, post-MI. ( N ) Quantification of EdU + proliferating single artery cells post-MI. p=0.0186. Scale bars: B , 50µm; D , H , 500µm; E , F , I , J , 200 µm; K - M , 50 µm. trans, transient; cyc, cycling; P, postnatal day; EC, endothelial cell; Tam, Tamoxifen; MI, myocardial infarction; lig, ligation; 1°, primary; LCA, left coronary artery; CM, cardiomyocyte; saEC, single artery endothelial cell

Article Snippet: Following mouse lines, Cx40CreER , ApjCreER , Cdk1 fl/fl (The Jackson Laboratory, 129S(B6N)-Cdk1 tm1Eddy /J, stock number 028028), Fzd4 fl/fl (The Jackson Laboratory, B6;129-Fzd4 tm2.1Nat /J, stock number 011078), Wls fl/fl (The Jackson Laboratory, Wls tm1.1Whsu /J, stock number 027484), Rosa26 TdTomato Cre reporter line (The Jackson Laboratory, B6.Cg-Gt[ROSA]26Sortm9[CAG-TdTomato]Hze/J, stock number 007909), Cx40 eGFP/+ and Axin2-d2EGFP were used in the experiments.

Techniques: Expressing, Control, Knock-Out, Ligation

Journal: bioRxiv

Article Title: Cell-autonomous Wnt activity promotes transient re-programming and cell cycle re-entry of coronary artery endothelial cells

doi: 10.64898/2026.02.23.707374

Figure Lengend Snippet: ( A ) Secondary alone control for immunostaining shown in . ( B ) Schematic showing positions of coronary occlusion through ligation of main LCA or primary branches of LCA to generate severe or moderate myocardial infarctions, respectively. ( C , D ) Confocal images of watershed regions from Fzd4 L/L ; Cx40CreER; Rosa26 TdTomato hearts with severe MI. Arrowheads point to TdTomato + lineage traced endothelial cells with non-EC morphologies. Brackets point to cluster of TdTomato + endothelial cells. ( E - G ) Confocal images of TdTomato + endothelial cells traced from pre-existing arteries showing non-EC morphology post-severe MI, in Fzd4 L/L ; Cx40CreER; Rosa26 TdTomato hearts. * point to TdTomato-lineage traced cells with cardiomyocyte like features. Scale bars: A , E , F , G , 50µm; C , D , 200µm. 1°, primary; 2°, secondary; LCA, left coronary artery; RCA, right coronary artery; EC, endothelial cell; MI, myocardial infarction; Ab, Antibody.

Article Snippet: Following mouse lines, Cx40CreER , ApjCreER , Cdk1 fl/fl (The Jackson Laboratory, 129S(B6N)-Cdk1 tm1Eddy /J, stock number 028028), Fzd4 fl/fl (The Jackson Laboratory, B6;129-Fzd4 tm2.1Nat /J, stock number 011078), Wls fl/fl (The Jackson Laboratory, Wls tm1.1Whsu /J, stock number 027484), Rosa26 TdTomato Cre reporter line (The Jackson Laboratory, B6.Cg-Gt[ROSA]26Sortm9[CAG-TdTomato]Hze/J, stock number 007909), Cx40 eGFP/+ and Axin2-d2EGFP were used in the experiments.

Techniques: Control, Immunostaining, Ligation

Journal: bioRxiv

Article Title: Cell-autonomous Wnt activity promotes transient re-programming and cell cycle re-entry of coronary artery endothelial cells

doi: 10.64898/2026.02.23.707374

Figure Lengend Snippet: ( A ) Experimental design to assess expression of VegfR2 and endomucin in single artery endothelial cells (saECs), post-MI. ( B , C ) Quantification of ( B ) VegfR2 and ( C ) Endomucin, in saECs, post-MI. p<0.0001. Each data point is a TdTomato + single artery cell. N=3 and N=5 hearts were quantified for VegfR2 and Endomucin expression, respectively. ( D ) Confocal images of saECs in Cx40CreER; Rosa26 TdTomato ; Cx40 eGFP/+ hearts, post-MI, showing TdTomato + saECs in red and expression of eGFP as a reporter for Cx40 expression in green. Arrowheads point to TdTomato + saECs with low levels of eGFP signal, as compared to the artery tip. ( E , H ) Confocal images of post-MI watersheds, showing TdTomato + saECs in red and immunostaining for VegfR2 in green in ( E ) control ( Cx40CreER; Rosa26 TdTomato ) and ( F ) knockout ( Fzd4 L/L ; Cx40CreER; Rosa26 TdTomato ) MI hearts. F , G are insets from E and show TdTomato + saECs express VegfR2 (orange arrowheads). I , J are insets from H and show reduced expression of VegfR2 by TdTomato + saECs (magenta arrowheads). ( K , N ) Confocal images of post-MI watersheds, showing TdTomato + saECs in red and immunostaining for Endomucin in green in ( K ) control ( Cx40CreER; Rosa26 TdTomato ) and ( N ) knockout ( Fzd4 L/L ; Cx40CreER; Rosa26 TdTomato ) MI hearts. L , M are insets from K and show TdTomato + saECs express Endomucin (orange arrowheads). O , P are insets from N and show reduced expression of Endomucin by TdTomato + saECs (magenta arrowheads). Scalebars: 50µm. saECs, single artery endothelial cells; P, postnatal day; KO, knockout; cap, capillary; MI, myocardial infarction.

Article Snippet: Following mouse lines, Cx40CreER , ApjCreER , Cdk1 fl/fl (The Jackson Laboratory, 129S(B6N)-Cdk1 tm1Eddy /J, stock number 028028), Fzd4 fl/fl (The Jackson Laboratory, B6;129-Fzd4 tm2.1Nat /J, stock number 011078), Wls fl/fl (The Jackson Laboratory, Wls tm1.1Whsu /J, stock number 027484), Rosa26 TdTomato Cre reporter line (The Jackson Laboratory, B6.Cg-Gt[ROSA]26Sortm9[CAG-TdTomato]Hze/J, stock number 007909), Cx40 eGFP/+ and Axin2-d2EGFP were used in the experiments.

Techniques: Expressing, Immunostaining, Control, Knock-Out

Journal: Advanced Science

Article Title: Brd4 BD1 Domain Antagonism of MS436 Preserves Blood‐Brain Barrier Integrity via Rnf43/β‐Catenin Signaling Pathway

doi: 10.1002/advs.202515584

Figure Lengend Snippet: Rnf43 regulates the level of TJ through Fzd4. A) Immunofluorescent staining for Zo‐1 with bEnd.3 lacking Fzd4, Fzd6 or Fzd10. Scale bar, 50 µm. B) WB detection for TJ proteins and β‐catenin with bEnd.3 lacking Fzd4, Fzd6 or Fzd10. C) Statistic analysis of relative intensity of TJ proteins and β‐catenin in WB detection, n=4. D) Co‐IP detection of Rnf43 and Fzd4. E) Co‐location detection of Rnf43 and Fzd4. Scale bar, 10 µm. F) WB detection for Fzd4 after Rnf43 knockdown. G) Statistic analysis of relative intensity of Fzd4 in WB detection, n=4. H) Detection of Fzd4 after Rnf43 knockdown. Scale bar, 10 µm. I) Statistic analysis of relative signal intensity of Fzd4, n=4. Data were presented as mean ± SEM, one‐way ANOVA, Two‐tailed Student's t ‐test, ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies and dilutions were used for immunostaining and western blotting: Brd4 (Abcam ab128874, Rabbit, 1:200); Brd4 (Invitrogen A301‐985A‐T, Rabbit, 1:500); Zo‐1 (Invitrogen 40‐2200, Rabbit, 1:500); Zo‐1 (Proteintech 21773‐1‐AP, Rabbit, 1:500); Claudin 5 (Invitrogen 352 500, Mouse, 1:500); Claudin 5 (Abclonal A10207, Rabbit, 1:500); GFAP (Sigma, G6171, Mouse, 1:1000); β‐catenin (Cell Signaling Technology, 8480s, Rabbit, 1:500); Fzd4 (Saribio, K109620P, Rabbit, 1:1 k); biotinylated IsolectinB4 (Vector Laboratories, B‐1205, 1:600); HA (Cell Signaling Technology, 3724s, Rabbit, 1:1 k); Flag (Sigma, F7425, Mouse, 1:2 k); β‐actin (Proteintech, 20536‐1‐AP, Rabbit, 1:10 000); β‐actin (Proteintech; 60008‐1‐Ig Mouse,1:2000); IgG (Bioss, bs‐0295p; Rabbit, 1:1 k).

Techniques: Staining, Co-Immunoprecipitation Assay, Knockdown, Two Tailed Test

Journal: Advanced Science

Article Title: Brd4 BD1 Domain Antagonism of MS436 Preserves Blood‐Brain Barrier Integrity via Rnf43/β‐Catenin Signaling Pathway

doi: 10.1002/advs.202515584

Figure Lengend Snippet: Brd4 regulates the expression of TJ proteins depends on its BD1 domain. A) Regulation of Claudin 5 by BD domains. Scale bar, 10 µm. B) Immunofluorescent staining for Zo‐1 with bEnd.3 overexpressing BD1 and BD2. Scale bar, 20 µm. C) Statistic analysis of relative intensity of Zo‐1, n=4. D) WB detection for TJ proteins with bEnd.3 overexpressing BD1 or BD2. E) Statistic analysis of relative intensity of TJ proteins in WB detection, n=4. F) Regulation of Claudin 5 by Brd4 mutants. Scale bar, 10 µm. G) Detection of the regulation of Brd4 mutants on the expression of Rnf43. H) Detection of Fzd4 after mutation of Brd4 . Scale bar, 10 µm. Data were presented as mean ± SEM, one‐way ANOVA, Two‐tailed Student's t ‐test, ns, no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies and dilutions were used for immunostaining and western blotting: Brd4 (Abcam ab128874, Rabbit, 1:200); Brd4 (Invitrogen A301‐985A‐T, Rabbit, 1:500); Zo‐1 (Invitrogen 40‐2200, Rabbit, 1:500); Zo‐1 (Proteintech 21773‐1‐AP, Rabbit, 1:500); Claudin 5 (Invitrogen 352 500, Mouse, 1:500); Claudin 5 (Abclonal A10207, Rabbit, 1:500); GFAP (Sigma, G6171, Mouse, 1:1000); β‐catenin (Cell Signaling Technology, 8480s, Rabbit, 1:500); Fzd4 (Saribio, K109620P, Rabbit, 1:1 k); biotinylated IsolectinB4 (Vector Laboratories, B‐1205, 1:600); HA (Cell Signaling Technology, 3724s, Rabbit, 1:1 k); Flag (Sigma, F7425, Mouse, 1:2 k); β‐actin (Proteintech, 20536‐1‐AP, Rabbit, 1:10 000); β‐actin (Proteintech; 60008‐1‐Ig Mouse,1:2000); IgG (Bioss, bs‐0295p; Rabbit, 1:1 k).

Techniques: Expressing, Staining, Mutagenesis, Two Tailed Test