Journal: PLoS ONE

Article Title: Role of integrin alpha8 in murine model of lung fibrosis

doi: 10.1371/journal.pone.0197937

Figure Lengend Snippet: (A) Western blot showing the absence of ITGA8 expression in Cre+ cells compared to Cre- cells. (B) Adhesion assay showing decreased binding to the substrates nephronectin (NPNT) and fibronectin (FN) by Cre+ cells. (C-G) PDGFRβ-selected cells were treated with TGFβ (10 ng/ml) or vehicle. mRNA expression is presented as fold change relative to Cre- in the untreated group (vehicle). Genes include: Itga8 (C), Col1a1 (D), Acta2 (E), Ctgf (F), and Fn1 (G). (* p <0.05, mean±SEM, n = 4).

Article Snippet: 96-well plates were coated with 1 μg/ml recombinant mouse nephronectin (R&D Systems) or rat plasma fibronectin (Sigma-Aldrich) at 4°C.

Techniques: Western Blot, Expressing, Cell Adhesion Assay, Binding Assay

Journal: eLife

Article Title: Nephronectin-integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

doi: 10.7554/eLife.74307

Figure Lengend Snippet:

Article Snippet: Nunc Lab-tek II 8-well chamber slides (Sigma) were coated with 1.5 μg/cm 2 with poly- d -lysine (MP Biomedicals) for 1 hr at room temperature, followed by recombinant mouse or human Npnt (R&D Systems) at 1.5 μg/cm 2 for 2 hr at 37°C.

Techniques: Transfection, Construct, shRNA, Virus, In Vitro, Recombinant, Plasmid Preparation, PCR Cloning, Staining

Journal: bioRxiv

Article Title: Nephronectin-Integrin α8 signaling is required for proper migration of periocular neural crest cells during chick corneal development

doi: 10.1101/2021.10.13.464255

Figure Lengend Snippet: (A) Cross-section of E5 eye immunostained for Npnt and Fn. Npnt appears in an increasing gradient from the periocular region into the cornea, whereas Fn stains both the periocular mesenchyme and cornea. (B) All pNC respond to Fn via expression of α5β1, but a subpopulation of pNC that reside in the region adjacent to the presumptive cornea express both α5β1 and α8β1, and become competent to also read the additional gradient of Npnt in the ECM, thus migrating into the corneal region. Abbreviations: pNC, periocular neural crest; oc, optic cup; ps, primary stroma; en, corneal endothelium.

Article Snippet: Samples were sectioned at 10 μm, rehydrated, then immunostained with Npnt antibody (1:100; orb221700, Biorbyt) following standard procedures.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

doi: 10.1038/srep45181

Figure Lengend Snippet: ( A ) qRT-PCR analysis of Npnt expression in teeth, skin, lungs, livers, kidney, heart, eyes, and brains of E14.5 embryos (n = 3). Gapdh was used as the internal control. *P < 0.05. Error bars represent mean ± S.D. ( B ) qRT-PCR analysis of Npnt expression in teeth obtained from mice on E11, E13, E14, E15, E16, E18, P0, P3, and P7. Gapdh was used as the internal control. ( C ) Npnt (green) and collagen IV (red) expressions in E11, E13, and E14 teeth, as detected by immunohistochemistry. Nuclei were stained with DAPI (blue). Scale bars: 50 μm. L; lingual side, B; buccal side.

Article Snippet: Immunohistochemistry was performed using primary antibodies to Npnt (1:500, R&D System), Sox2 (1:250, abcam), Ambn (1:500, Santa Cruz), Ki67 (1:250, Cell Signaling), and Collagen IV (1:500, Millipore) for 16 hours at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Control, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

doi: 10.1038/srep45181

Figure Lengend Snippet: ( A ) Expressions of Npnt (green) and Sox2 (red) in representative E14 molar and incisor, as detected by immunohistochemistry. Nuclei were stained with DAPI (blue). ( B ) Expressions of Npnt (green) and Sox2 (red) (upper panels), Npnt (green) and Ki67 (red) (middle panels), and Npnt (green) and Ambn (red) (lower panels) in representative P1 incisors, as detected by immunohistochemistry. Nuclei were stained with DAPI (blue). Arrowheads show area of Npnt expression, which was decreased in Sox2+ dental epithelial cells in the adjacent cervical loop. Scale bars: 50 μm. L; lingual side, B; buccal side, La; labial side.

Article Snippet: Immunohistochemistry was performed using primary antibodies to Npnt (1:500, R&D System), Sox2 (1:250, abcam), Ambn (1:500, Santa Cruz), Ki67 (1:250, Cell Signaling), and Collagen IV (1:500, Millipore) for 16 hours at 4 °C.

Techniques: Immunohistochemistry, Staining, Expressing

Journal: Scientific Reports

Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

doi: 10.1038/srep45181

Figure Lengend Snippet: ( A ) Two-day organ cultures of representative E13 tooth germs transfected with control or Npnt siRNA. Npnt (green) and Sox2 (red) expressions were detected by immunohistochemistry. Nuclei were stained with DAPI (blue). ( B ) Schematic representation of cultured tooth germs. ( C ) Ratio of Sox2+ cells in cultured tooth germs divided into lingual and buccal sides following transfection with control siRNA or Npnt siRNA. ( D ) Sox2 (green) expression in M3H1 cells cultured in dishes with or without recombinant Npnt coating. Nuclei were stained with DAPI (blue). ( E ) Ratio of Sox2+ cells among M3H1 cells cultured with or without Npnt coating. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. *P < 0.05. Error bars represent mean ± S.D. Scale bars: 50 μm.

Article Snippet: Immunohistochemistry was performed using primary antibodies to Npnt (1:500, R&D System), Sox2 (1:250, abcam), Ambn (1:500, Santa Cruz), Ki67 (1:250, Cell Signaling), and Collagen IV (1:500, Millipore) for 16 hours at 4 °C.

Techniques: Transfection, Control, Immunohistochemistry, Staining, Cell Culture, Expressing, Recombinant

Journal: Scientific Reports

Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

doi: 10.1038/srep45181

Figure Lengend Snippet: ( A ) E13 tooth germs were transfected with control or Npnt siRNA and cultured for 8 days. ( B ) Relative tooth size plot (n = 10), with average tooth germ size in control siRNA group set at 1.0. ( C ) Cell proliferation was analyzed using a CCK-8 assay after coating the dishes with recombinant Npnt or transfection with Npnt. BrdU incorporation of M3H1 cells after coating dishes with recombinant Npnt or transfection with Npnt. The ratio was calculated as BrdU-positive cells/DAPI-stained nuclei. ( D ) Western blotting results of cyclin D1, Sox2, V5, and Gapdh in M3H1 cells transfected with mock vector or Npnt expression vector. Gapdh was used as the internal control. The full-length blots are presented in . *P < 0.05. Error bars represent mean ± S.D. Scale bars: 100 μm.

Article Snippet: Immunohistochemistry was performed using primary antibodies to Npnt (1:500, R&D System), Sox2 (1:250, abcam), Ambn (1:500, Santa Cruz), Ki67 (1:250, Cell Signaling), and Collagen IV (1:500, Millipore) for 16 hours at 4 °C.

Techniques: Transfection, Control, Cell Culture, CCK-8 Assay, Recombinant, BrdU Incorporation Assay, Staining, Western Blot, Plasmid Preparation, Expressing

Journal: Scientific Reports

Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

doi: 10.1038/srep45181

Figure Lengend Snippet: ( A ) Schematic representation of Npnt constructs. Npnt-FL, full-length Npnt; Npnt-ΔEGF, Npnt with deletion of EGF-like repeats domain; Npnt-ΔRGD, Npnt with deletion of RGD and MAM domains. ( B ) Ratio of Sox2+ cells among M3H1 cells in dishes coated with Npnt, collagen I, collagen IV, laminin I, and fibronectin. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( C ) Cell proliferation was analyzed using a CCK-8 assay after transfection with Npnt-FL, Npnt-ΔEGF, or Npnt-ΔRGD. BrdU incorporation analysis after transfection with Npnt-FL, Npnt-ΔEGF, or Npnt-ΔRGD. The ratio was calculated as BrdU-positive cells/DAPI-stained nuclei. ( D ) Ratio of Sox2+ positive cells among M3H1 cells transfected with Npnt-FL, Npnt-ΔEGF, or Npnt-ΔRGD. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. *P < 0.05. Error bars represent mean ± S.D.

Article Snippet: Immunohistochemistry was performed using primary antibodies to Npnt (1:500, R&D System), Sox2 (1:250, abcam), Ambn (1:500, Santa Cruz), Ki67 (1:250, Cell Signaling), and Collagen IV (1:500, Millipore) for 16 hours at 4 °C.

Techniques: Construct, Staining, CCK-8 Assay, Transfection, BrdU Incorporation Assay

Journal: Scientific Reports

Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

doi: 10.1038/srep45181

Figure Lengend Snippet: ( A ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( B ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, with or without EGFR siRNA. Gapdh was used as the internal control. ( C ) Ratio of Sox2 cells among M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. The ratio was calculated as Sox2+ cells/DAPI-stained nuclei. ( D ) qRT-PCR analysis of Sox2 expression in M3H1 cells transfected with Npnt-FL, or treated with EGFR inhibitor gefitinib or lapatinib. Gapdh was used as the internal control. *P < 0.05. Error bars represent mean ± S.D.

Article Snippet: Immunohistochemistry was performed using primary antibodies to Npnt (1:500, R&D System), Sox2 (1:250, abcam), Ambn (1:500, Santa Cruz), Ki67 (1:250, Cell Signaling), and Collagen IV (1:500, Millipore) for 16 hours at 4 °C.

Techniques: Transfection, Staining, Quantitative RT-PCR, Expressing, Control

Journal: Scientific Reports

Article Title: Nephronectin plays critical roles in Sox2 expression and proliferation in dental epithelial stem cells via EGF-like repeat domains

doi: 10.1038/srep45181

Figure Lengend Snippet: ( A ) Western blotting of P-Akt, Akt, PI3K, P-PLCγ, P-Stat5, P-Erk1/2, Erk1/2, and Gapdh in M3H1 cells transfected with Npnt-FL or Npnt-ΔEGF, and cultured for 48 hours. Gapdh was used as the internal control. The full-length blots are presented in . ( B ) Western blotting of P-Akt and Akt in M3H1 cells with or without recombinant Npnt treatment for 60 minutes. Gapdh was used as the internal control. The full-length blots are presented in . ( C ) Ratio of Sox2+ cells among M3H1 cells transfected with Npnt-FL and treated with different doses of PI3K inhibitor (LY294002). The ratio was calculated as Sox2 positive cells/DAPI-stained nuclei. ( D ) BrdU incorporation in M3H1 cells treated with different doses of LY294002. The ratio was calculated as BrdU-positive cells/DAPI-stained nuclei. *P < 0.05. Error bars represent mean ± S.D.

Article Snippet: Immunohistochemistry was performed using primary antibodies to Npnt (1:500, R&D System), Sox2 (1:250, abcam), Ambn (1:500, Santa Cruz), Ki67 (1:250, Cell Signaling), and Collagen IV (1:500, Millipore) for 16 hours at 4 °C.

Techniques: Western Blot, Transfection, Cell Culture, Control, Recombinant, Staining, BrdU Incorporation Assay