Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) RT-qPCR was performed during osteoblast differentiation using primers specific for ( A ) NPNT, ( B ) ALP, ( C ) Col1A1, and ( D ) OCN. Gene expression was normalized to 18S and compared to the expression at day 0. ( E ) NPNT expression construct (pcDNA3.1-NPNT-c-myc/His), which encodes a mouse full length NPNT, was generated. ( F ) Detection of NPNT protein in the conditioned medium of COS-7 cells transfected with NPNT expression construct using anti-c-myc antibody. ( G ) NPNT was detected in osteoblast supernatants using anti-NPNT antibody. HtrA1 was used as positive marker for osteoblast differentiation and β-Actin was used as loading control. Western blot images are presented as cropped format. Full length blots are presented in . *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Quantitative RT-PCR, Gene Expression, Expressing, Construct, Generated, Transfection, Marker, Control, Western Blot

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Tibias from sham group (left) and OVX group (right) were visualized by μCT scanning, and the ROI focused on the trabecular region below the growth plate for quantitative analysis of ( B ) Tb.BV/TV, ( C ) Tb.Th, ( D ) Tb.N and ( E ) Tb.Sp. Real-time PCR quantitative analysis of ( F ) Runx2, ( G ) OCN and ( H ) NPNT gene expression in mouse forelimbs. n = 6/group. ( I ) μCT images of trabecular bone in the region between head and neck of femur from patients with osteoarthritis (top) and osteoporosis (bottom), and quantitative analysis of ( J ) Tb.BV/TV, ( K ) Tb.Th, ( L ) Tb.N and ( M ) Tb.Sp. Real-time PCR quantitative analysis of ( N ) Runx2, ( O ) OCN and ( P ) NPNT gene expression in human femora. n = 20/group. The gene expressions were normalized to β-Actin and 18S. *P < 0.05.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Representative microscopic images of H&E and immunostaining using NPNT and CD31-specific antibody on tibia sections of sham and OVX mice. Quantitative analysis of ( B ) NPNT and ( C ) CD31 protein expressions in the tibias of OVX mice. *P < 0.05, **P < 0.01.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Immunostaining

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Representative microscopic views of scratch wound healing assays performed using SVEC cells treated with recombinant mouse NPNT (500 ng/ml) from 0 to 16 hours. Scale bar, 100 μm. ( B ) Quantitative analysis of cell migration area. ( C ) Representative images showing tube-like structure formation by SVEC cells following treatment with recombinant mouse NPNT (500 ng/ml) for 24 hours. Scale bar, 100 μm. ( D , E ) Quantitative analysis of branch points and tube lengths. PBS and bFGF were used as a negative and positive control respectively. ( F ) Representative images showing that recombinant mouse NPNT (200 ng/ml) induced vessel outgrowth from metatarsals dissected from E17.5 embryos. Scale bar, 250 μm. ( G ) Quantitative analysis of vessel sprouting. PBS and VEGF (50 ng/ml) were used as a negative and positive control respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Recombinant, Migration, Positive Control

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Western blot images showing the treatment of SVEC cells by mouse recombinant NPNT resulted in the phosphorylation of ERK1/2 and p-38, but not Akt. β-Actin was used as a loading control. ( B – D ) Quantification of signal intensities of p-ERK1/2, p-p38 and p-Akt by ImageJ. Induction ratios at each timepoint were compared to 0 minute, with p-ERK1/2 normalized to ERK1/2, p-p38 normalized to p-38 and p-Akt normalized to Akt. Western blot images are presented as cropped format. Full length blots are presented in . *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Western Blot, Recombinant, Phospho-proteomics, Control

Journal: Scientific Reports

Article Title: NPNT is Expressed by Osteoblasts and Mediates Angiogenesis via the Activation of Extracellular Signal-regulated Kinase

doi: 10.1038/srep36210

Figure Lengend Snippet: ( A ) Representative microscopic images of scratch wound healing assays showing NPNT-induced endothelial cell migration was blocked in the presence of U0126 (5 μM). Scale bar, 100 μm. Quantitative analyses showing that ( B ) NPNT-induced endothelial cell migration and ( C ) tube-like structure formation were significantly inhibited by U0126. PBS and bFGF were used as a negative and positive control, respectively. ( D ) Representative images showing that NPNT-induced angiogenesis was inhibited in the presence of U0126 (5 μM). Scale bar, 250 μm. ( E ) Quantitative analysis of vessel sprouting. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Recombinant mouse NPNT (R&D Systems), human bFGF (PeproTech, Inc.), human VEGF (PeproTech, Inc.) and ERK1/2 inhibitor U0126 (Promega) were purchased for the assays.

Techniques: Migration, Positive Control

Journal: Scientific Reports

Article Title: Nephronectin promotes breast cancer brain metastatic colonization via its integrin-binding domains

doi: 10.1038/s41598-020-69242-1

Figure Lengend Snippet: The integrin-binding sites of NPNT are important for adhesion. ( a ) bEND.3 cells were seeded onto rmNPNT- or pluronic coated surfaces for 0, 1, 3, 5 or 24 h. Cell lysates were harvested and analysed by Western blotting for tyrosine phosphorylation. ( b ) bEND.3 cells were seeded onto rmNPNT- or Pluronic coated surfaces for 3 h. Harvested cell lysates were analysed for specific tyrosine phosphorylated proteins using the Proteome Profiler antibody array (human Phospho-kinase Array). ( c ) Adhesion of 66cl4-EV cells to rmNPNT was analysed by real-time cell analysis using the xCELLigence system. The experiments were performed at least twice with similar results and with two technical replicates per run. Impedance created by adhering cells gave the arbitrary “cell index” value that is proportional to the amount of adhered cells. Adhesion was recorded every 15 min for 12 h. Wells were coated with either 3% BSA as a negative control or 10 μg purified wild-type rmNPNT (Wild-type) or rmNPNT mutated in the integrin-binding sites (Mutant). Error bars show the pooled standard deviation. ( d ) Wells were coated with 10 μg purified wild-type rmNPNT. Adhesion of 66cl4-EV cells in the presence of RGD-blocking peptide or scrambled negative control peptide. p = 0.019. ( e ) 66cl4 cells were seeded onto a confluent monolayer of MBECs. After three hours, non-adherent cells were washed off and adherent cells were counted. Fold differences between the cells is shown in the graph (N = 2, n = 3). p = 0.013. ( f ) 66cl4 cells were seeded onto a confluent monolayer of bEND.3 cells. After three hours, non-adherent cells were washed off and adherent cells were counted. Fold differences between the cells is shown in the graph (N = 3, n = 3). p = NS. ( g ) 66cl4 cells were seeded onto a confluent monolayer of MBECs. Transmigration of 66cl4 cells was followed by imaging every 5 min for 24 h. Every cell that transmigrated through the monolayer was recorded and fold differences between the cells is presented in the graph (N = 3, n = 3), p = 0.044.

Article Snippet: E-Plates were coated with either purchased rmNPNT (Cat: 4,298-NP-050, RD Systems, Minneapolis, MN) or purified wild-type rmNPNT (wt) or mutated rmNPNT (mutant).

Techniques: Binding Assay, Western Blot, Phospho-proteomics, Ab Array, Cell Analysis, Negative Control, Purification, Mutagenesis, Standard Deviation, Blocking Assay, Transmigration Assay, Imaging

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: 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: ( A ) Cross-section of embryonic day (E)5 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. 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: 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: Samples were sectioned at 10 μm, rehydrated, then immunostained with Npnt antibody (1:100; orb221700, Biorbyt) following standard procedures.

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

Journal: Cells

Article Title: A Tight Control of Non-Canonical TGF-β Pathways and MicroRNAs Downregulates Nephronectin in Podocytes

doi: 10.3390/cells11010149

Figure Lengend Snippet: Non-canonical pathways predominantly mediate post-transcriptional regulation of NPNT under baseline conditions. ( a , b ) qPCR for NPNT mRNA in cultured human podocytes, untreated and after treatment with inhibitors of the non-canonical pathways ( a ), PD169316, SP600125, PD98059, AKT inhibitor, MG-132, and Y-27632, or with SM16 and/or SIS3 for canonical pathway inhibition ( b ). NPNT mRNA expression normalized to HPRT is given as the change compared to untreated cells. n = 6–7 independent experiments; ** p < 0.01. ( c , d ) NPNT protein expression in cultured human podocytes, untreated and after treatment with different inhibitors of the non-canonical ( c ) and the canonical pathways ( d ) as described in ( a , b ). NPNT protein expression, normalized to GAPDH, is given as the change compared to untreated cells. A representative Western blot result is shown for each treatment. n = 6–11 independent experiments; n.s. = not significant, * p < 0.05. ( e , f ) Human podocytes were treated as described in ( a , b ). NPNT protein expression was measured by ELISA in the cell culture supernatant and plotted as the change compared to untreated controls. n = 3 independent experiments.

Article Snippet: Measurement of the NPNT protein concentration in the cell culture supernatant of podocytes was performed with the human Nephronectin ELISA kit (MyBioSource, San Diego, CA, USA), according to the manufacturers’ protocol.

Techniques: Cell Culture, Inhibition, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay