nephronectin  (R&D Systems)

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

Journal: FEBS Open Bio

Article Title: Nephronectin mediates p38 MAPK ‐induced cell viability via its integrin‐binding enhancer motif

doi: 10.1002/2211-5463.12544

Figure Lengend Snippet: Cell surface distribution of NPNT in 66cl4 cells. (A) Immunofluorescence microscopy showing extracellular NPNT detected on 66cl4 cells expressing wild‐type NPNT and 66cl4‐ EV cells when preincubated with rm NPNT for 1 h prior fixing. 66cl4‐ EV was used as a negative control. Detection of collagen V on 66cl4‐ NPNT cells was used as a positive control. Primary antibodies were visualized with Alexa Fluor 488. Nucleus is stained blue with Hoechst. Scale bar 10 μm. (B) Z profile comparing the green and blue channels was calculated by normalizing mean intensity per slice in the stack for each channel using the image of 66cl4 cells overexpressing NPNT shown above. (C) Brightfield microscopy images of 66cl4‐ EV cells growing on uncoated plates ( EV ) in contrast to rm NPNT ‐coated plates ( EV rm NPNT ) at 24 h.

Article Snippet: The effect of incubating 66cl4‐EV cells with 2 μg·mL −1 recombinant mouse NPNT (rmNPNT) (R&D systems, Minneapolis, MN, USA; Cat: 4298‐NP‐050) in PBS for 1 h prior fixing was also investigated.

Techniques: Immunofluorescence, Microscopy, Expressing, Negative Control, Positive Control, Staining

Journal: FEBS Open Bio

Article Title: Nephronectin mediates p38 MAPK ‐induced cell viability via its integrin‐binding enhancer motif

doi: 10.1002/2211-5463.12544

Figure Lengend Snippet: Reverse‐phase protein array analysis of NPNT ‐mediated signaling. The Venn diagram includes number of proteins significantly regulated and/or modified ( P < 0. 05) in all four biological replicates. (A) The pink circle in the Venn diagram, ‘ NPNT vs EV ’, denotes the log‐fold change values triggered in 66cl4‐ NPNT cells in comparison with 66cl4‐ EV cells. The blue circle, ‘ EV rm NPNT vs EV ’, represents 66cl4‐ EV cells cultured on rm NPNT ( EV rm NPNT ) in comparison with 66cl4‐ EV cells seeded in noncoated wells. The purple circle represents proteins regulated by the integrin‐binding motifs of NPNT ; the effect of a single mutation in the RGD motif ( RGD → RGE ) versus mutations in both RGD and EIE motifs ( RGD ‐ EIE ‐> RGE ‐ AIA ). (B) Box plot showing log2 protein abundance of the four overlapping proteins from the Venn diagram.

Article Snippet: The effect of incubating 66cl4‐EV cells with 2 μg·mL −1 recombinant mouse NPNT (rmNPNT) (R&D systems, Minneapolis, MN, USA; Cat: 4298‐NP‐050) in PBS for 1 h prior fixing was also investigated.

Techniques: Protein Array, Modification, Comparison, Cell Culture, Binding Assay, Mutagenesis, Quantitative Proteomics

Journal: FEBS Open Bio

Article Title: Nephronectin mediates p38 MAPK ‐induced cell viability via its integrin‐binding enhancer motif

doi: 10.1002/2211-5463.12544

Figure Lengend Snippet: Top predicted molecular and cellular functions. RPPA results from RGE vs RGE‐AIA group were analyzed using the web‐based software application ingenuity pathway analysis (IPA) tool to identify the most significant NPNT‐responsive functions

Article Snippet: The effect of incubating 66cl4‐EV cells with 2 μg·mL −1 recombinant mouse NPNT (rmNPNT) (R&D systems, Minneapolis, MN, USA; Cat: 4298‐NP‐050) in PBS for 1 h prior fixing was also investigated.

Techniques: Software, Activation Assay

Journal: FEBS Open Bio

Article Title: Nephronectin mediates p38 MAPK ‐induced cell viability via its integrin‐binding enhancer motif

doi: 10.1002/2211-5463.12544

Figure Lengend Snippet: Nephronectin mediates cell viability via p38 signaling pathways. (A) Indicated variants of 66cl4 cells were treated with (±) 4 μ m p38 MAPK inhibitor ( BIRB 796) for 24 h, in addition to serum deprivation. Where indicated, 66cl4‐ EV cells were stimulated by adding 2 μg·mL −1 rm NPNT to the cell culture medium. Cell viability was determined using CellTiter‐Glo. (B) Viability of NPNT expressing, 4T1 cells with a NPNT ‐targeted short hairpin (sh‐ NPNT ) and a nontargeting sh RNA (sh‐ctr) was tested using CellTiter‐Glo. Significance is tested using a two‐tailed Student's t‐test. * P < 0. 05, ** P < 0. 005, *** P < 0. 0001. Error bars represent SD . N = number of independent experiments, n = total number of replicates in each test group. (C) Illustration summarizing the cellular effects of integrin binding to wild‐type or mutated NPNT via p38 MAPK .

Article Snippet: The effect of incubating 66cl4‐EV cells with 2 μg·mL −1 recombinant mouse NPNT (rmNPNT) (R&D systems, Minneapolis, MN, USA; Cat: 4298‐NP‐050) in PBS for 1 h prior fixing was also investigated.

Techniques: Protein-Protein interactions, Cell Culture, Expressing, Two Tailed Test, Binding Assay