mr npnt  (R&D Systems)

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: 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

Journal: FEBS Open Bio

Article Title: Antibodies against nephronectin ameliorate anti‐type II collagen‐induced arthritis in mice

doi: 10.1002/2211-5463.12758

Figure Lengend Snippet: Real‐time PCR primer sequences.

Article Snippet: Mouse nephronectin (Npnt) protein [called Npnt (R&D) protein in this study] was from R&D systems (Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: FEBS Open Bio

Article Title: Antibodies against nephronectin ameliorate anti‐type II collagen‐induced arthritis in mice

doi: 10.1002/2211-5463.12758

Figure Lengend Snippet: Primer sequences for cloning of Npnt.

Article Snippet: Mouse nephronectin (Npnt) protein [called Npnt (R&D) protein in this study] was from R&D systems (Minneapolis, MN, USA).

Techniques: Cloning, Sequencing

Journal: FEBS Open Bio

Article Title: Antibodies against nephronectin ameliorate anti‐type II collagen‐induced arthritis in mice

doi: 10.1002/2211-5463.12758

Figure Lengend Snippet: Expression of Npnt and α8 integrin in human and mouse tissues. Npnt (A) and α8 integrin (B) expression in healthy tissues were evaluated by real‐time PCR. Data are representative of three or more independent experiments with similar results. Data are presented as means ± SEM.

Article Snippet: Mouse nephronectin (Npnt) protein [called Npnt (R&D) protein in this study] was from R&D systems (Minneapolis, MN, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: FEBS Open Bio

Article Title: Antibodies against nephronectin ameliorate anti‐type II collagen‐induced arthritis in mice

doi: 10.1002/2211-5463.12758

Figure Lengend Snippet: Establishing the sandwich ELISA system for Npnt. (A) Schematic representation of Npnt and its truncated variants. Cell binding sequences (RGD sequences) in the linker segment and the epitope in the MAM domain of anti‐Npnt3 antibody are indicated. (B) Specificity of Npnt3 and 20D1a antibodies by western blotting. (C) Diagram (top panel) and standard curve (lower panel) of the ELISA. Recombinant mNpnt (R&D) protein was used as the standard. (D) Expression of Npnt in cell lines by RT–PCR. Human or mouse cell lines were left or right panel, respectively. (E) Detection of Npnt in supernatants from cell lines used in (C). Data are representative of three or more independent experiments with similar results. Data are presented as means ± SEM.

Article Snippet: Mouse nephronectin (Npnt) protein [called Npnt (R&D) protein in this study] was from R&D systems (Minneapolis, MN, USA).

Techniques: Sandwich ELISA, Binding Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: FEBS Open Bio

Article Title: Antibodies against nephronectin ameliorate anti‐type II collagen‐induced arthritis in mice

doi: 10.1002/2211-5463.12758

Figure Lengend Snippet: Npnt in plasma and urine samples. (A) Npnt in plasma and urine samples from healthy mice and humans. (B) Npnt in plasma samples from various murine autoimmune disease models. Plasma samples were tested on the day when inflammation was exacerbated as follows, for ConA‐hepatitis, 24 h after ConA injection; for CAIA, 7 days after LPS injection; for EAE, 14 days after MOG or PLP peptide immunization. Data are representative of three or more independent experiments with similar results. * P < 0.05 or ** P < 0.01 versus plasma samples from wild‐type mice (Student's t ‐test).

Article Snippet: Mouse nephronectin (Npnt) protein [called Npnt (R&D) protein in this study] was from R&D systems (Minneapolis, MN, USA).

Techniques: Clinical Proteomics, Injection

Journal: FEBS Open Bio

Article Title: Antibodies against nephronectin ameliorate anti‐type II collagen‐induced arthritis in mice

doi: 10.1002/2211-5463.12758

Figure Lengend Snippet: Generation of neutralizing antibodies for Npnt. (A) Cross‐reactivity of anti‐Npnt‐FD antibody to plates coated with Npnt, fibronectin, or GRGDS peptide‐coupled with BSA (1 μg·mL −1 ) by direct ELISA. (B) Specificity of anti‐Npnt‐FD antibody by western blotting. (C, D) NIH3T3 cells were allowed to adhere to 96‐well plates precoated with full‐length of Npnt or Npnt‐Link (C), fibronectin, or vitronectin (D) in the presence of indicated concentrations of anti‐Npnt‐FD antibody. Normal rabbit IgG was used as a control antibody. Data are presented as mean ± SEM. Data are representative of three or more independent experiments with similar results. * P < 0.05, anti‐Npnt‐FD antibody versus control antibody (Student's t ‐test).

Article Snippet: Mouse nephronectin (Npnt) protein [called Npnt (R&D) protein in this study] was from R&D systems (Minneapolis, MN, USA).

Techniques: Direct ELISA, Western Blot, Control

Journal: FEBS Open Bio

Article Title: Antibodies against nephronectin ameliorate anti‐type II collagen‐induced arthritis in mice

doi: 10.1002/2211-5463.12758

Figure Lengend Snippet: Npnt neutralization by anti‐Npnt‐FD antibody ameliorates CAIA. (A) Protocol for CAIA with anti‐Npnt‐FD antibody treatment. The antibody was administered to BALB/c mice on days −4 and 0 relative to LPS injection during CAIA induction. (B) Npnt expression in arthritic joints on day 6, synovial fibroblasts, or synovial macrophages were evaluated by real‐time PCR. * P < 0.05, ** P < 0.01, wild‐type versus CAIA (Student's t ‐test). (C) Arthritis scores of arthritic mice treated with control antibody (rabbit IgG) or an anti‐Npnt‐FD antibody at the indicated time points ( n = 5 per group). * P < 0.05, ** P < 0.01, anti‐Npnt‐FD antibody versus control Ig (Student's t ‐test). Data are presented as means ± SEM. (D) Representative images of the forepaw and hindpaw on day 7 are shown. (E–G) Representative histology of normal joints and arthritic joints on day 14 from mice treated with control antibody or anti‐Npnt‐FD antibody. Sections were stained with HE (E, G) or Safranin‐O (F). Magnified views of the boxed areas a and b are shown in F and G, respectively. Scale bar = 2mm (E), 200μm (F and G). Data are representative of three or more independent experiments with similar results.

Article Snippet: Mouse nephronectin (Npnt) protein [called Npnt (R&D) protein in this study] was from R&D systems (Minneapolis, MN, USA).

Techniques: Neutralization, Injection, Expressing, Real-time Polymerase Chain Reaction, 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: 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

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: Patient and Tumor Characteristics, and Risk of Death from Breast Cancer According to NPNT Positive Staining Pattern

Article Snippet: For adhesion assays E-plates (Cat.: 05469830001) were coated with recombinant mouse NPNT (Cat.: 4298-NP-050, R&D Systems, Minneapolis, NE) at 0.4, 2, or 10 μg/ml for 4 hours at 37°C prior to seeding.

Techniques: Staining, Biomarker Discovery

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: Granular NPNT staining patterns are associated with poor outcome in breast cancer patients. Representative images of IHC- and HES-stained TMA samples showing (A) different staining patterns: no staining, nuclear staining, diffuse cytoplasmic staining, and single cells positive or granular cytoplasmic staining using antibodies towards human NPNT (Atlas Antibodies/Sigma) imaged at 600× magnification. The images show representative cases of each staining phenotype, and the cases shown are Luminal A, Luminal A, Luminal B (HER2−), Luminal A and Luminal B (HER2−), respectively. Scale bars: 50 μm. Survival analysis showing association between no granular cytoplasmic staining (red line), 1-10% granular positive cells (thick dotted blue line), or >10% granular positive cells (solid black line) on (B) cumulative risk of death from breast cancer and (C) overall survival for all 842 patients represented in the TMAs.

Article Snippet: For adhesion assays E-plates (Cat.: 05469830001) were coated with recombinant mouse NPNT (Cat.: 4298-NP-050, R&D Systems, Minneapolis, NE) at 0.4, 2, or 10 μg/ml for 4 hours at 37°C prior to seeding.

Techniques: Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: Phenotypically different patterns of NPNT staining in MMTV-PyMT transgenic mouse tumors. (A) Representative images of IHC- and HES-stained MMTV-PyMT primary tumors. NPNT was detected using antibodies towards mouse NPNT (Atlas Antibodies/Sigma). Figure shows antibody-stained sections, IgG Isotype control, control without antibodies (-ab), and HES-stained section from the same region. (B) Representative images of IHC-stained MMTV-PyMT lung metastases. Arrows indicate no staining (open arrow), diffuse cytoplasmic staining (filled arrow), and granular staining (triangle). Images are representative of a series of samples from nine mice.

Article Snippet: For adhesion assays E-plates (Cat.: 05469830001) were coated with recombinant mouse NPNT (Cat.: 4298-NP-050, R&D Systems, Minneapolis, NE) at 0.4, 2, or 10 μg/ml for 4 hours at 37°C prior to seeding.

Techniques: Staining, Transgenic Assay, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: NPNT is located in granules in 66cl4-NPNT primary tumors. Representative images of IHC-stained 66cl4-EV and 66cl4-NPNT primary tumors using V5-specific antibodies (Cell Signaling Technology). The left and middle panels show antibody-stained sections, whereas the right panels show rabbit IgG Isotype staining controls. Granular staining is indicated with open triangles. Images are representative of a series of samples from five mice.

Article Snippet: For adhesion assays E-plates (Cat.: 05469830001) were coated with recombinant mouse NPNT (Cat.: 4298-NP-050, R&D Systems, Minneapolis, NE) at 0.4, 2, or 10 μg/ml for 4 hours at 37°C prior to seeding.

Techniques: Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: NPNT increases adhesion and anchorage-independent growth. (A) Adhesion of 4T1-shFF and 4T1-shNPNT cells in an xCELLigence adhesion assay. The figure shows rescue of adhesion in 4T1-shNPNT cells in plates coated with 2 μg/ml recombinant mouse NPNT (rmNPNT). 4T1-shFF cells express a nontargeting control shRNA. Data are presented as mean cell index ± 95% CI from two experiments ( n =4), 1 hour after seeding. (B) xCELLigence adhesion assay of 66cl4-cells expressing either EV or NPNT wild-type, NPNT RGE, or NPNT RGE-AIA mutants in uncoated plates. Data are presented as mean cell index ± 95%CI from four experiments ( n =3-12), 1 hour after seeding. Lower panel shows Western blot of cell lysates confirming NPNT overexpression detected with the anti-V5 antibody. The image is cropped to display only relevant bands, but the full blot is shown in A . (C) Adhesion assay of 66cl4-EV cells in plates coated with rmNPNT at 0.4 μg/ml, 2 μg/ml, or 10 μg/ml prior to seeding. Data are presented as mean cell index ± 95% CI from two experiments (n=4), 1 hour after seeding. (D) Adhesion assay of 66cl4-EV cells in rmNPNT-coated wells in presence of either scrambled peptide or RGD-blocking peptide. Data are presented as mean cell index±SEM from three experiments with n =2 in each experiment. (E) Migration assay in xCELLigence CIM-plates using 66cl4-cells expressing either EV or NPNT wild-type, NPNT RGE, or NPNT RGE-AIA mutants. Migration towards 10% FCS was recorded every 5 minutes, and data are presented as mean cell index ± 95%CI at 12 hours from 5 individual experiments with n =2-4 technical replicates in each experiment. (F) Cell proliferation assay with 1.3×10 6 cells per 25-cm 2 flask. Cells were harvested and counted after 24, 48, and 72 hours, and data are shown as mean ± SD from three individual experiments. (G) Soft agar assay for anchorage-independent growth of 66cl4-cells expressing EV, NPNT, NPNT RGE, or NPNT RGE-AIA. Two thousand cells/well in 12-well plates were seeded in 0.36% agarose/α-MEM medium containing 10% FCS and grown for 10 days. Data presented as mean ± 95% CI from 3 individual experiments ( n =3-9). (H) Clonogenic cell survival assay with minimal seeding density of 66cl4-cells expressing EV, NPNT, NPNT RGE, or NPNT RGE-AIA. A total of 10 cells/well were seeded in 12-well plates and grown as single colonies for 10 days. Graph shows number of colonies as mean ±95%CI from five individual experiments with n =3-9 replicates per experiment. # indicates P =.064 when comparing EV-cells with NPNT-expressing cells. All in vitro data except the qRT-PCR and the proliferation assay were analyzed with linear regression models. *** P <.0001, ** P <.005, * P <.05. NPNT increases adhesion and anchorage-independent growth. (A) Adhesion of 4T1-shFF and 4T1-shNPNT cells in an xCELLigence adhesion assay. The figure shows rescue of adhesion in 4T1-shNPNT cells in plates coated with 2 μg/ml recombinant mouse NPNT (rmNPNT). 4T1-shFF cells express a nontargeting control shRNA. Data are presented as mean cell index ± 95% CI from two experiments ( n =4), 1 hour after seeding. (B) xCELLigence adhesion assay of 66cl4-cells expressing either EV or NPNT wild-type, NPNT RGE, or NPNT RGE-AIA mutants in uncoated plates. Data are presented as mean cell index ± 95%CI from four experiments ( n =3-12), 1 hour after seeding. Lower panel shows Western blot of cell lysates confirming NPNT overexpression detected with the anti-V5 antibody. The image is cropped to display only relevant bands, but the full blot is shown in Supplementary Figure S3 A . (C) Adhesion assay of 66cl4-EV cells in plates coated with rmNPNT at 0.4 μg/ml, 2 μg/ml, or 10 μg/ml prior to seeding. Data are presented as mean cell index ± 95% CI from two experiments (n=4), 1 hour after seeding. (D) Adhesion assay of 66cl4-EV cells in rmNPNT-coated wells in presence of either scrambled peptide or RGD-blocking peptide. Data are presented as mean cell index±SEM from three experiments with n =2 in each experiment. (E) Migration assay in xCELLigence CIM-plates using 66cl4-cells expressing either EV or NPNT wild-type, NPNT RGE, or NPNT RGE-AIA mutants. Migration towards 10% FCS was recorded every 5 minutes, and data are presented as mean cell index ± 95%CI at 12 hours from 5 individual experiments with n =2-4 technical replicates in each experiment. (F) Cell proliferation assay with 1.3×10 6 cells per 25-cm 2 flask. Cells were harvested and counted after 24, 48, and 72 hours, and data are shown as mean ± SD from three individual experiments. (G) Soft agar assay for anchorage-independent growth of 66cl4-cells expressing EV, NPNT, NPNT RGE, or NPNT RGE-AIA. Two thousand cells/well in 12-well plates were seeded in 0.36% agarose/α-MEM medium containing 10% FCS and grown for 10 days. Data presented as mean ± 95% CI from 3 individual experiments ( n =3-9). (H) Clonogenic cell survival assay with minimal seeding density of 66cl4-cells expressing EV, NPNT, NPNT RGE, or NPNT RGE-AIA. A total of 10 cells/well were seeded in 12-well plates and grown as single colonies for 10 days. Graph shows number of colonies as mean ±95% CI from five individual experiments with n =3-9 replicates per experiment. # indicates P =.064 when comparing EV-cells with NPNT-expressing cells. All in vitro data except the qRT-PCR and the proliferation assay were analyzed with linear regression models. *** P <.0001, ** P <.005, * P <.05.

Article Snippet: For adhesion assays E-plates (Cat.: 05469830001) were coated with recombinant mouse NPNT (Cat.: 4298-NP-050, R&D Systems, Minneapolis, NE) at 0.4, 2, or 10 μg/ml for 4 hours at 37°C prior to seeding.

Techniques: Cell Adhesion Assay, Recombinant, Control, shRNA, Expressing, Western Blot, Over Expression, Blocking Assay, Migration, Proliferation Assay, Soft Agar Assay, Clonogenic Cell Survival Assay, In Vitro, Quantitative RT-PCR

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: NPNT promotes colonization of the lung via its integrin-binding motifs. 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA mutants were injected into the lateral tail vein to assess tumor cell colonization of the lung using 24-25 mice per group. (A) Lungs were collected after 3 weeks and weighed (mean±SD). ** P <.0005 when analyzed by two-tailed t test. The data have been merged from two separate experiments using 14-15 and 9-10 mice per group. (B) RTB assay using genomic DNA from whole lung lysates as template ( n =24-25). Graphs show mean RTB ± SD, *** P <.0001 when analyzed by two-tailed t-test. (C) Images of three representative lungs from each group of mice. Macroscopic view of representative lung lobes imaged to detect mCherry positive tumor cells (bottom row). The tissues were imaged using the EVOS FL Auto Cell Imaging System with an inverted microscope and a Sony ICX445 monochrome CCD camera and visualized using 4× objectives. Each image was created using a stitch of several images to cover the entire lung lobe. (D) Representative images of IHC-stained lungs from mice using NPNT antibodies (Atlas Antibodies/Sigma). The top and middle rows show antibody-stained sections at two different magnifications, whereas the lower row shows rabbit IgG isotype staining controls. Scale bars: top and lower row: 100 μm, middle row: 10 μm. Tu: indicates tumor area, Lu: indicates normal lung tissue. Images are representative of a series of samples from five mice.

Article Snippet: For adhesion assays E-plates (Cat.: 05469830001) were coated with recombinant mouse NPNT (Cat.: 4298-NP-050, R&D Systems, Minneapolis, NE) at 0.4, 2, or 10 μg/ml for 4 hours at 37°C prior to seeding.

Techniques: Binding Assay, Expressing, Injection, Two Tailed Test, Imaging, Inverted Microscopy, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: Nephronectin is Correlated with Poor Prognosis in Breast Cancer and Promotes Metastasis via its Integrin-Binding Motifs

doi: 10.1016/j.neo.2018.02.008

Figure Lengend Snippet: NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in . IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm NPNT is localized in extracellular vesicles. (A) Western blot of isolated microvesicles and exosomes from 66cl4-cells expressing EV, NPNT wild-type, NPNT RGE, or NPNT RGE-AIA detected with anti-V5 antibodies (CST). ALIX and CHMP4B were used as markers for microvesicles and exosomes, GM130 as a negative control and GAPDH for normalization control. Whole-cell lysates of 66cl4-NPNT cells were included as control. The images are cropped to display only relevant bands. Full-length blots are shown in Supplementary Figure S3. IF showing colocalization between the exosomal marker CHMP4B and NPNT detected with anti-CHMP4B and NPNT antibodies (Abnova) and visualized with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies, respectively, of (B) lung samples from in vivo lung colonization assay and (C) MMTV-PyMT mammary tumor samples. Images are representative of a series from five mice. Nuclei were stained with Hoechst. -ab reflects a control with no primary antibody. Scale bar; 5 μm

Article Snippet: For adhesion assays E-plates (Cat.: 05469830001) were coated with recombinant mouse NPNT (Cat.: 4298-NP-050, R&D Systems, Minneapolis, NE) at 0.4, 2, or 10 μg/ml for 4 hours at 37°C prior to seeding.

Techniques: Western Blot, Isolation, Expressing, Negative Control, Control, Marker, In Vivo, Staining