mr npnt  (R&D Systems)

Journal: PLoS ONE

Article Title: Endotoxin-induced acute lung injury in mice with postnatal deletion of nephronectin

doi: 10.1371/journal.pone.0268398

Figure Lengend Snippet: (A) qPCR analysis of Npnt mRNA levels in lung and kidneys from Cre- and Cre+ mice after 4 weeks on the tamoxifen diet followed by normal chow. Expression is shown relative to Cre-. Shown are the means (bars) ± SE. Immunofluorescence staining for NPNT (red) in Cre- (B) and Cre+ (C) lungs from mice fed a tamoxifen diet for 4 weeks followed by chow. Nuclei were stained with DAPI. The insets show the localization of NPNT in Cre- and reduced staining in Cre+ lung tissue. Representative hematoxylin and eosin staining of sections of lung from uninjured Cre- (D) and Cre+ (E) using the same time course of dietary treatment. Scale bar = 50 μm for all images.

Article Snippet: Sections were incubated overnight at 4°C with a goat anti-mouse NPNT polyclonal (R&D, catalog number AF4298) or IgG isotype control (Jackson Immunoresearch, catalog number 005-000-003) at 2 μg/ml in TBST containing 1% BSA.

Techniques: Expressing, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: Endotoxin-induced acute lung injury in mice with postnatal deletion of nephronectin

doi: 10.1371/journal.pone.0268398

Figure Lengend Snippet: (A) Schematic of the time course of LPS injury and sample acquisition. (B) mRNA levels of Npnt in lungs from Cre- mice were measured by qPCR and expression calculated relative to uninjured. qPCR was also used to determine if expression of integrin α8 ( Itga8 ) (C) or the NPNT homologue EGFL6 (D) is altered by LPS injury of Cre+ lungs. Gene expression was calculated relative to uninjured Cre-. Shown are the medians (bars) with 95% confidence intervals. Data were analyzed using either the Mann-Whitney or Kruskal-Wallis statistical test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. (E-H) Immunofluorescence for NPNT at day 3 (E and F) and day 7 (G and H) post LPS in Cre- (E and G) and Cre+ (F and H) lung tissue. Nuclei were stained with DAPI (blue). Scale bar = 100 μm.

Article Snippet: Sections were incubated overnight at 4°C with a goat anti-mouse NPNT polyclonal (R&D, catalog number AF4298) or IgG isotype control (Jackson Immunoresearch, catalog number 005-000-003) at 2 μg/ml in TBST containing 1% BSA.

Techniques: Expressing, Gene Expression, MANN-WHITNEY, Immunofluorescence, 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:

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: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: NPNT gene overexpression by adeno-associated virus (serotype 2/9) in mouse hearts upregulated NPNT and attenuated cardiac dysfunction post-MI. (A-B) NPNT was transiently expressed in mice heart both at protein and mRNA level. NPNT was expressed in P1 (neonate mice, n = 2 for WB, n = 6 for qPCR) but hardly detected in P56 (adult mice, n = 2 for WB, n = 4 for qPCR), * P < 0.05. (C) Protocol of AAV2/9-mediated NPNT overexpression and time points for tissue collection in adult male C57BL/6 mice. (D) GFP-expressing AAV was imaged in the myocardium (n = 2). (E) , (F) The protein and mRNA levels of NPNT in the infarct border zone were all significantly upregulated on 3 weeks post-injection (n = 3). * P < 0.05. Parameters of LVID-d, LVID-s (G) , EF and FS (H) detected on day 1 and day 28 showing that MI surgery caused equal decreased heart function among the three MI group. (n = 8-10). * P < 0.05 versus sham group, and # P < 0.05 versus MI-PBS group. Scale bar, 100 μm.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: Over Expression, Virus, Expressing, Injection

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: NPNT limited myocardial infarction size, fibrosis, and improved survival after MI. (A) Representative images of tetrazolium chloride staining at day 28 post-MI in the mice hearts from the four groups. Ratios of infarcted area vs. left ventricular tissue area are calculated in the graph (n = 6). Scale bar, 1 mm. (B) Masson trichrome staining of infarcted mouse myocardium in different groups on day 28 after MI within infarct zone, border zone, remote zone (n = 5). (C) Ratios of fibrosis area vs. total cross section area are analysed using Image J. (D) Survival analysis within 28 days showed significantly lower mortality in the MI-NPNT group compared with other two MI groups. ( P = 0.0197) (n = 18). # P < 0.05 versus MI-PBS group. Scale bar, 100 μm.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: Staining

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: NPNT promoted angiogenesis in infarcted mouse hearts. CD31 and α-SMA were immunofluorescent stained in infarct border zone at 4 weeks post-MI. (A) Representative images and quantitative analysis of myocardial capillary density determined by the number of endothelial-specific CD31-positive cells per mm 2 (n = 6). (B) Representative images and quantification of newly formed vessels analysed by α-SMA-positive arterioles per field of view (FOV) (n = 6). * P < 0.05 versus sham group. # P < 0.05 versus MI-PBS group. Scale bar, 200 µm.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: Staining

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: NPNT induced endothelial cell wound healing and tube formation in vitro. (A) Representative images and statistical analysis of the cell migration area of scratch wound healing assay in PBS, rm-NPNT (recombinant mouse NPNT, 500 ng/ml, same dose in following experiments unless indicated) and rh-bFGF (recombinant human bFGF, 50 ng/ml, same dose in following experiments unless indicated) treated HUVEC for 12h. Scale bar, 500 µm. (B) Representative images and quantification of migrated cells in transwell assay for 24h. Scale bar, 100 µm. (C) Representative images of tube-formation assay in HUVEC seeded on Matrigel (upper). Branching points and tube formatted were shown using Image J (lower). PBS, rm-NPNT and rh-bFGF was added into the culture medium, respectively. Quantification of branching points and tube length was calculated in 4 different fields. * P < 0.05. Scale bar, 500 µm.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: In Vitro, Migration, Wound Healing Assay, Recombinant, Transwell Assay, Tube Formation Assay

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: NPNT activated the EGFR/JAK2/STAT3 signalling pathway in HUVECs. (A-C) Recombinant mouse NPNT protein was added to HUVECs (500 ng/ml) over time (0 min, 5 min, 10 min, 20 min, 30 min, 60 min). The p-EGFR/EGFR ratio, p-JAK2/JAK2 ratio and p-STAT3/STAT3 were increased at 5 min, peaked at 20 min and decreased thereafter. Each bar represents the mean ± SD of three independent experiments. * P < 0.05 versus control group.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: Recombinant, Control

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: Gefitinib and C188-9 abrogated NPNT-induced EGFR/JAK2/STAT3 signalling activation. HUVECs were preincubated with Gefitinib (EGFR inhibitor, 10 μM) or C188-9 (STAT3 inhibitor, 10 μM) for 1 h and then stimulated with NPNT (500 ng/ml) for 20 min. The phosphorylation levels of EGFR, JAK2 and STAT3 were all decreased in Gefitinib pretreatment. While only phosphorylated STAT3 not phosphorylated EGFR or phosphorylated JAK2 was inhibited by C188-9 treatment, indicating NPNT activated EGFR followed by activating JAK2/STAT3 pathway. Gefitinib and C188-9 were dissolved with DMSO, thus identical dose of DMSO was added to the control group and NPNT group, respectively. * P < 0.05 versus DMSO group, # P < 0.05 versus NPNT group.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: Activation Assay, Phospho-proteomics, Control

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: Gefitinib and C188-9 impaired NPNT-induced endothelial cell migration and tube formation in vitro. (A) Representative images and quantification of scratch wound healing assays (Scale bar, 500 μm), (B) Transwell assay (scale bar, 100 μm) and (C) tube formation assay (Scale bar, 500 μm) indicating that NPNT-promoted endothelial cell migration and tube formation were significantly inhibited by Gefitinib (10 μM) or C188-9 (10 μM). Quantitative analysis of NPNT-induced cell migration and tube formation. * P < 0.05.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: Migration, In Vitro, Transwell Assay, Tube Formation Assay

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: Gefitinib and C188-9 impaired Adv-NPNT-induced endothelial cell migration in vitro. (A) Representative images and quantification of scratch wound healing assays (Scale bar, 500 μm) and (B) Transwell assay indicating that Adv-NPNT-promoted endothelial cell migration and were significantly inhibited by Gefitinib (10 μM) or C188-9 (10 μM). * P < 0.05. Scale bar, 100 μm.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: Migration, In Vitro, Transwell Assay

Journal: International Journal of Medical Sciences

Article Title: Nephronectin promotes cardiac repair post myocardial infarction via activating EGFR/JAK2/STAT3 pathway

doi: 10.7150/ijms.71780

Figure Lengend Snippet: NPNT activated EGFR/JAK2/STAT3 signalling pathway in vivo. The phosphorylation levels of EGFR and its downstream factors JAK2, STAT3 were higher in the MI group mice hearts and further increased in the MI-NPNT group on day 28 post-MI. Each bar represents the mean ± SD of n = 4-6 mice in each group. * P < 0.05 versus sham group, # P < 0.05 versus MI-PBS group.

Article Snippet: Recombinant mouse NPNT (R&D, 500 ng/ml) or human bFGF (Pepro Tech, Inc., 50 ng/ml) protein were used in serum-free medium instead of complete medium.

Techniques: In Vivo, Phospho-proteomics