Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Meclozine attenuates the MAPK pathway of FGF2-treated tibiae in the organ culture system. (A) Upper panels: Representative images of E16.5 tibiae of wild-type mice after 4-day culture. Scale bares indicate 1 mm. Lower panels: Absolute bone length after 4-day treatment. Dots indicate the length. Lines are drawn between dots of the same individual. Statistical significance was analyzed by paired Student’s t -test. (B) Enrichment plots of two MAPK signaling-associated gene sets identified by GSEA between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. (C) Heatmap depicting the expression of the genes in REACTOME_MAPK_FAMILY_SIGNALING_CASCADES signature and ST_P38_MAPK_PATHWAY signature between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. (D) Enrichment plots of BMP signaling-associated gene set identified by GSEA between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. (E) Heatmap depicting the expression of the Ihh , Bmp2 , Bmp4 , and Bmp7 between FGF2- and FGF2+, and FGF2+ and FGF2+ meclozine in the articular cartilage of ex vivo cultured tibiae. MAPK, mitogen-activated protein kinase; GSEA, Gene set enrichment analysis.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Organ Culture, Ex Vivo, Cell Culture, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Treatment protocol of FGF2 and meclozine are determined for evaluating vertebral ossification in larval zebrafish. (A) Treatment regimen of FGF2 for larval zebrafish. (B) Quantification of ossified vertebrae after FGF2 treatment. Dots indicate the number of ossified vertebrae of each sample, and bars indicate means. Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD. (C) Representative images of larval zebrafish from the lateral view at seven dpf stained with Alizarin red after 30 ng/mL FGF2 treatment from eight hpf to seven dpf. Arrows: ossified vertebrae. Scale bar indicates 500 µm. (D) Survival curve of larval zebrafish treated with each dose of meclozine from eight hpf to seven dpf.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Meclozine attenuates spinal and craniofacial bone ossification in FGF2-treated larval zebrafish. (A) Representative images of larval zebrafish from the lateral view at seven dpf stained with Alizarin red after 30 ng/mL FGF2 treatment, with or without 1 µM meclozine, from eight hpf to seven dpf. Arrows: ossified vertebrae. Scale bar indicates 500 µm. (B) Quantification of ossified vertebrae after FGF2 treatment, with or without meclozine. Dots indicate the number of ossified vertebrae of each sample, and bars indicate means. Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD. hpf, hours post-fertilization; dpf, days post-fertilization. (C) Representative craniofacial bone elements of larval zebrafish from anteroposterior view at seven dpf stained with Alizarin red after FGF2 treatment, with or without meclozine. Scale bar indicates 500 µm. (D) Quantification of the number of each ossified craniofacial bone element, including ceratohyal (ch), hyomandibular (hm), branchiostegal ray (br), dentary (d), entopterygoid (en), maxilla (m), and opercle (o), after FGF2 treatment with or without meclozine. Data values are presented as means and standard deviation (SD). Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Staining, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Meclozine Attenuates the MARK Pathway in Mammalian Chondrocytes and Ameliorates FGF2-Induced Bone Hyperossification in Larval Zebrafish

doi: 10.3389/fcell.2021.694018

Figure Lengend Snippet: Meclozine ameliorates FGF2-induced hyper ossification in larval zebrafish. (A) Representative craniofacial cartilage elements of larval zebrafish from ventral view, three-dimensional (3D) view, and single layer at seven dpf in Tg (col2a1a:EGFP) after FGF2 treatment, with or without meclozine. Scale bar indicates 100 µm. (B) Quantification of area of craniofacial cartilage, including ceratohyal (ch), hyosymplectic (h), and palatoquadrate (pq) after FGF2 treatment with or without meclozine. (C) Representative craniofacial cartilage and bone elements of larval zebrafish from ventral view, 3D view, and single layer at seven dpf in Tg (col2a1a:EGFP) stained with Alizarine red after FGF2 treatment, with or without meclozine. Scale bar indicates 100 µm. (D) Quantification of relative ossification area, including ceratohyal (ch), hyomandibular (hm), and quadrate (q) after FGF2 treatment with or without meclozine. Relative ossification area was calculated by dividing each red signal area by each green area. Data values are presented as means and standard deviation (SD). Statistical significance was analyzed by one-way ANOVA with post-hoc Tukey HSD.

Article Snippet: FGF2 (3339-FB-025, R and D Systems) was administered in the presence or absence of 20 μM meclozine (155341, MP Biomedicals) for 4 days.

Techniques: Staining, Standard Deviation

Journal:

Article Title: Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

doi: 10.1096/fj.09-134056

Figure Lengend Snippet: VEGF-A, FGF-2 and VEGF-C-induced corneal angiogenesis and lymphangiogenesis in C57BL/6 and BALB/c mice. A) Pellets containing VEGF-A (200 ng), FGF-2 (100 ng), VEGF-C (400 ng), or vehicle control (PBS) were implanted into corneas of C57BL/6 and BALB/c mice. Corneal angiogenesis and lymphangiogenesis were examined 6 d after implantation. B) Double staining of corneal flat mounts for angiogenic (CD31, green) and lymphangiogenic (LYVE-1, red) endothelium. Scale bar = 400 μm. C, D) Quantitative analysis of angiogenesis (C) and lymphangiogenesis (D) in PBS-, VEGF-A-, FGF-2-, or VEGF-C-implanted corneas on d 6 (n=5–11). E) Quantitation of the number of lymphatic tips in corneas of VEGF-A-, FGF-2-, VEGF-C-, or vehicle control-implanted eyes (n=3–13). *P < 0.05; **P < 0.01.

Article Snippet: Matrigel plug assay Mice were injected subcutaneously with 0.2 ml Matrigel (356230; BD Biosciences) containing PBS, 1 μg FGF-2 (3139-FB; R&D Systems), or 2 μg VEGF-C (2179-VC; R&D Systems).

Techniques: Double Staining, Quantitation Assay

Journal:

Article Title: Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

doi: 10.1096/fj.09-134056

Figure Lengend Snippet: Corneal lymphangiogenesis and conjunctival lymphatic vessels. Corneal flatmounts with lymphangiogenesis were examined on d 6 after implantation and divided into two groups, with or without preexisting lymphatic vessels in the conjunctiva. A) Double staining of corneal flatmounts for angiogenesis (CD31, green) and lymphangiogenesis (LYVE-1, red) with or without lymphatic tube structures in VEGF-A-implanted BALB/c mice. Blood vessels (arrowheads) and lymphatic vessels (arrows) in the conjunctiva are indicated. B) Frequency of the corneas with LYVE-1+ lymphatic tube structures in the pellet-implanted site of C57BL/6 and BALB/c mice. C) Quantitative analysis of lymphangiogenesis (white) and angiogenesis (black) in VEGF-A-implanted BALB/c corneas on d 6 (n=4 and 7). D) Photographs and immunohistochemistry of CD31 and LYVE-1 with or without conjunctival removal. E, F) Corneal angiogenesis and lymphangiogenesis were examined 6 d after PBS or VEGF-A implantation with or without conjunctival removal. Arrows indicate lymphatic hyperplasia around the excised area. G, H) Quantitative analysis of lymphangiogenesis (G) and angiogenesis (H) in PBS- or VEGF-A-implanted corneas on d 6 with or without conjunctival removal (n=4–7). I, J) Correlation between lymphangiogenic area (d 6) and limbus lymphatic ratio in VEGF-A (I) or FGF-2 implantation (J) (n=6–11). *P < 0.05; **P < 0.01. Scale bars = 400 μm.

Article Snippet: Matrigel plug assay Mice were injected subcutaneously with 0.2 ml Matrigel (356230; BD Biosciences) containing PBS, 1 μg FGF-2 (3139-FB; R&D Systems), or 2 μg VEGF-C (2179-VC; R&D Systems).

Techniques: Double Staining, Immunohistochemistry

Journal:

Article Title: Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains

doi: 10.1096/fj.09-134056

Figure Lengend Snippet: GF-induced angiogenesis and lymphangiogenesis in Matrigel plug assay. A) Double staining for angiogenic (CD31, green) and lymphatic endothelium (LYVE-1, red) of PBS-, FGF-2-, or VEGF-C-containing Matrigel in C57BL/6 and BALB/c mice, 10 d after subcutaneous injection of Matrigel. B, C) Quantitative analysis of LYVE-1+ area (red, B) and CD31+ area (green, C) in Matrigel (n=4). D) Double staining for lymphatic endothelium (podoplanin, red) and macrophages (CD11b, green) of Matrigel in C57BL/6 and BALB/c mice (d 10). E, F) Quantitative analysis of podoplanin+ area (red, E) and CD11b+ area (green, F) in Matrigel (n=4). G) Double staining for lymphatic endothelium (LYVE-1, red) and proliferating marker (Ki67, green) of FGF-2-containing Matrigel in BALB/c mice (d 10). H) Quantitative analysis of number of LYVE1+ Ki67+ cells in Matrigel (n=4). *P < 0.05; **P < 0.01. Scale bars = 100 μm (A, D); 50 μm (G).

Article Snippet: Matrigel plug assay Mice were injected subcutaneously with 0.2 ml Matrigel (356230; BD Biosciences) containing PBS, 1 μg FGF-2 (3139-FB; R&D Systems), or 2 μg VEGF-C (2179-VC; R&D Systems).

Techniques: Matrigel Assay, Double Staining, Injection, Marker