Journal: International Dental Journal

Article Title: Role of TGF-β3 in Regulating Neural Crest Cell Fate and Craniofacial Development: Insights From Zebrafish Models

doi: 10.1016/j.identj.2025.103874

Figure Lengend Snippet: Effects of tgfb3 knockdown on zebrafish craniofacial cartilage and bone formation. A, qPCR validation of tgfb3 mRNA reduction. B, Western blot confirming Tgfb3 knockdown efficiency. C, Morphological and statistical analysis of zebrafish after Tgfb3 knockdown (5 × magnification). D–E. Impaired cartilage formation assessed by Alcian blue staining and in situ hybridization. F-G, Disrupted bone formation visualized via Alizarin red staining and in situ hybridization ( P < .05, * P < .01).

Article Snippet: After blocking and washing, membranes were incubated overnight at 4 °C with primary antibodies (1:1000 unless noted) against: TGFB3, ALP, Runx2, OSX (Abcam); Smad2, pSmad2, Smad4 (CST); Col1 (Affinity); GAPDH (1:5000; Proteintech); and β-actin (1:5000; Origene).

Techniques: Knockdown, Biomarker Discovery, Western Blot, Staining, In Situ Hybridization

Journal: International Dental Journal

Article Title: Role of TGF-β3 in Regulating Neural Crest Cell Fate and Craniofacial Development: Insights From Zebrafish Models

doi: 10.1016/j.identj.2025.103874

Figure Lengend Snippet: Effects of Tgfb3 Knockdown on Neural Crest Cells in Zebrafish. A, Volcano plot of DEGs between the Control Morpholino (CMO) and the DMO)groups. Red dots represent upregulated genes, while blue dots indicate downregulated genes. B, GO enrichment analysis of the differentially expressed genes, with red boxes highlighting enriched entries in pathways related to neural crest cells. C, Heatmap showing expression levels of neural crest cell-related genes ( hoxb3a, hoxd3a, gbx2, dlx6a, gsc, b3gat1a, id2a, tfap2a, tfap2b, and otx2b ) in both groups. D, qPCR results for neural crest cell-related genes in embryos at different developmental stages. E. In situ hybridization analysis showing changes in the expression of neural crest markers ( foxd3 and sox9a ) and the pharyngeal mesoderm marker ( gsc ) (magnified 5 times) ( P < .05, * P < .01, ** P < .001).

Article Snippet: After blocking and washing, membranes were incubated overnight at 4 °C with primary antibodies (1:1000 unless noted) against: TGFB3, ALP, Runx2, OSX (Abcam); Smad2, pSmad2, Smad4 (CST); Col1 (Affinity); GAPDH (1:5000; Proteintech); and β-actin (1:5000; Origene).

Techniques: Knockdown, Control, Expressing, In Situ Hybridization, Marker

Journal: International Dental Journal

Article Title: Role of TGF-β3 in Regulating Neural Crest Cell Fate and Craniofacial Development: Insights From Zebrafish Models

doi: 10.1016/j.identj.2025.103874

Figure Lengend Snippet: Effects of Tgfb3 Knockdown on NCSC Proliferation, Apoptosis, and Migration. A, Observation of neural crest stem cell culture in vitro. B, Immunofluorescence identification of neural crest stem cells. C, Expression of NCSC-specific genes ( P75, Pax3, Snail, Hnk1 ) in passages P0 to P5. D, mRNA expression levels of Tgfb3 in NCSCs following knockdown. E, Protein expression levels of Tgfb3 in NCSCs following knockdown. F, Proliferation of NCSCs assessed by the CCK-8 assay. G, EdU incorporation assay after 48 hours to detect NCSC proliferation. H, Apoptosis levels of NCSCs detected by flow cytometry. I, Scratch assay evaluating NCSC migration at 0, 12, and 24 hours (magnified 10 times) ( P < .05, * P < .01, ** P < .001, ns P > .05).

Article Snippet: After blocking and washing, membranes were incubated overnight at 4 °C with primary antibodies (1:1000 unless noted) against: TGFB3, ALP, Runx2, OSX (Abcam); Smad2, pSmad2, Smad4 (CST); Col1 (Affinity); GAPDH (1:5000; Proteintech); and β-actin (1:5000; Origene).

Techniques: Knockdown, Migration, Stem Cell Culture, In Vitro, Immunofluorescence, Expressing, CCK-8 Assay, Flow Cytometry, Wound Healing Assay

Journal: International Dental Journal

Article Title: Role of TGF-β3 in Regulating Neural Crest Cell Fate and Craniofacial Development: Insights From Zebrafish Models

doi: 10.1016/j.identj.2025.103874

Figure Lengend Snippet: Tgfb3 knockdown impairs chondrogenic and osteogenic differentiation of NCSCs in vitro . A, Alcian blue staining after 21-day chondrogenic induction. B-C, Osteogenic differentiation markers (ALP: day 7; Alizarin red: day 21). D, Western blot analysis of osteogenic markers ( P < .05).

Article Snippet: After blocking and washing, membranes were incubated overnight at 4 °C with primary antibodies (1:1000 unless noted) against: TGFB3, ALP, Runx2, OSX (Abcam); Smad2, pSmad2, Smad4 (CST); Col1 (Affinity); GAPDH (1:5000; Proteintech); and β-actin (1:5000; Origene).

Techniques: Knockdown, In Vitro, Staining, Western Blot