human 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: 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: For human Npnt expression in human tissues, a human multiple tissue cDNA panel was used (Takara, Kusatsu, Japan).

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: For human Npnt expression in human tissues, a human multiple tissue cDNA panel was used (Takara, Kusatsu, Japan).

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: 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: For human Npnt expression in human tissues, a human multiple tissue cDNA panel was used (Takara, Kusatsu, Japan).

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

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: 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: 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.

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: 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.

Techniques: Staining, Transgenic 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: 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: 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.

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: 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.

Techniques: Cell Adhesion Assay, Recombinant, 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: 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.

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: 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.

Techniques: Western Blot, Isolation, Expressing, Negative Control, Marker, In Vivo, 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: 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: Figure S1 Optimization of the of anti-human NPNT (Atlas Antibodies/Sigma) antibody using normal human kidney and breast cancer tissues.

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: Figure S1 Optimization of the of anti-human NPNT (Atlas Antibodies/Sigma) antibody using normal human kidney and breast cancer tissues.

Techniques: Staining, Transgenic 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: 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: Figure S1 Optimization of the of anti-human NPNT (Atlas Antibodies/Sigma) antibody using normal human kidney and breast cancer tissues.

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: Figure S1 Optimization of the of anti-human NPNT (Atlas Antibodies/Sigma) antibody using normal human kidney and breast cancer tissues.

Techniques: Cell Adhesion Assay, Recombinant, 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: Figure S1 Optimization of the of anti-human NPNT (Atlas Antibodies/Sigma) antibody using normal human kidney and breast cancer tissues.

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: Figure S1 Optimization of the of anti-human NPNT (Atlas Antibodies/Sigma) antibody using normal human kidney and breast cancer tissues.

Techniques: Western Blot, Isolation, Expressing, Negative Control, Marker, In Vivo, 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: 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: Validation of specificity of the antibodies anti-human NPNT (Atlas Antibodies/Sigma and anti-mouse NPNT (Abnova) on MMTV-PyMT mouse tissues is shown in .

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: Validation of specificity of the antibodies anti-human NPNT (Atlas Antibodies/Sigma and anti-mouse NPNT (Abnova) on MMTV-PyMT mouse tissues is shown in .

Techniques: Staining, Transgenic 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: 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: Validation of specificity of the antibodies anti-human NPNT (Atlas Antibodies/Sigma and anti-mouse NPNT (Abnova) on MMTV-PyMT mouse tissues is shown in .

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: Validation of specificity of the antibodies anti-human NPNT (Atlas Antibodies/Sigma and anti-mouse NPNT (Abnova) on MMTV-PyMT mouse tissues is shown in .

Techniques: Cell Adhesion Assay, Recombinant, 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: Validation of specificity of the antibodies anti-human NPNT (Atlas Antibodies/Sigma and anti-mouse NPNT (Abnova) on MMTV-PyMT mouse tissues is shown in .

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: Validation of specificity of the antibodies anti-human NPNT (Atlas Antibodies/Sigma and anti-mouse NPNT (Abnova) on MMTV-PyMT mouse tissues is shown in .

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