netrin 1  (R&D Systems)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Recombinant Human Netrin 1 Protein CF
    Description:
    The Recombinant Human Netrin 1 Protein from R D Systems is derived from NS0 The Recombinant Human Netrin 1 Protein has been validated for the following applications Bioactivity
    Catalog Number:
    6419-n1-025/cf
    Price:
    329
    Applications:
    Bioactivity
    Purity:
    >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie« Blue Staining.
    Conjugate:
    Unconjugated
    Size:
    25 ug
    Category:
    Proteins and Enzymes
    Source:
    NS0-derived Recombinant Human Netrin-1 Protein
    Buy from Supplier


    Structured Review

    R&D Systems netrin 1
    Isoflurane inhibits growth cone collapse induced by <t>Netrin-1,</t> but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p
    The Recombinant Human Netrin 1 Protein from R D Systems is derived from NS0 The Recombinant Human Netrin 1 Protein has been validated for the following applications Bioactivity
    https://www.bioz.com/result/netrin 1/product/R&D Systems
    Average 99 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    netrin 1 - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism"

    Article Title: Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism

    Journal: Anesthesiology

    doi: 10.1097/ALN.0b013e318287b850

    Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p
    Figure Legend Snippet: Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p

    Techniques Used: Incubation

    2) Product Images from "Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism"

    Article Title: Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism

    Journal: Anesthesiology

    doi: 10.1097/ALN.0b013e318287b850

    Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p
    Figure Legend Snippet: Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p

    Techniques Used: Incubation

    3) Product Images from "Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism"

    Article Title: Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism

    Journal: Anesthesiology

    doi: 10.1097/ALN.0b013e318287b850

    Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p
    Figure Legend Snippet: Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p

    Techniques Used: Incubation

    4) Product Images from "Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism"

    Article Title: Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a ?-Aminobutyric Acid Type A Receptor Mechanism

    Journal: Anesthesiology

    doi: 10.1097/ALN.0b013e318287b850

    Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p
    Figure Legend Snippet: Isoflurane inhibits growth cone collapse induced by Netrin-1, but does not alter branching induced by Netrin-1 Isoflurane (2.4%) inhibits Netrin-1/Laminin induced axonal growth cone collapse, and the effect is blocked by picrotoxincotreatment (A). Isoflurane (1.2%) does not alter axon branching induced by Netrin-1. Representative examples of neurons and neurolucida tracings of their axon arbors are shown. (B) and (E) are control neurons incubated for 8h in carrier gas alone. (C) and (F) are neurons with Netrin-1 and carrier gas, showing the increase in branching induced by Netrin-1. (D) and (G) show axonal arbors of neurons cotreated with Netrin-1 and 1.2% isoflurane. An analysis of branch number shows that isoflurane treatment does not alter branching (H). n=1,418 axonal growth cones in 46 fields for (A). n=81 neurons for (H). *In (A) indicates p

    Techniques Used: Incubation

    5) Product Images from "Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton"

    Article Title: Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4963-03.2004

    Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p
    Figure Legend Snippet: Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p

    Techniques Used: Labeling, Staining

    Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.
    Figure Legend Snippet: Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.

    Techniques Used: Labeling, Droplet Countercurrent Chromatography, Concentration Assay

    Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.
    Figure Legend Snippet: Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.

    Techniques Used:

    Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.
    Figure Legend Snippet: Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.

    Techniques Used: Transferring

    Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p
    Figure Legend Snippet: Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p

    Techniques Used:

    6) Product Images from "Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton"

    Article Title: Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4963-03.2004

    Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p
    Figure Legend Snippet: Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p

    Techniques Used: Labeling, Staining

    Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.
    Figure Legend Snippet: Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.

    Techniques Used: Labeling, Droplet Countercurrent Chromatography, Concentration Assay

    Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.
    Figure Legend Snippet: Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.

    Techniques Used:

    Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.
    Figure Legend Snippet: Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.

    Techniques Used: Transferring

    Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p
    Figure Legend Snippet: Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p

    Techniques Used:

    7) Product Images from "Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton"

    Article Title: Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4963-03.2004

    Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p
    Figure Legend Snippet: Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p

    Techniques Used: Labeling, Staining

    Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.
    Figure Legend Snippet: Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.

    Techniques Used: Labeling, Droplet Countercurrent Chromatography, Concentration Assay

    Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.
    Figure Legend Snippet: Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.

    Techniques Used:

    Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.
    Figure Legend Snippet: Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.

    Techniques Used: Transferring

    Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p
    Figure Legend Snippet: Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p

    Techniques Used:

    8) Product Images from "Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton"

    Article Title: Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4963-03.2004

    Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p
    Figure Legend Snippet: Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p

    Techniques Used: Labeling, Staining

    Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.
    Figure Legend Snippet: Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.

    Techniques Used: Labeling, Droplet Countercurrent Chromatography, Concentration Assay

    Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.
    Figure Legend Snippet: Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.

    Techniques Used:

    Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.
    Figure Legend Snippet: Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.

    Techniques Used: Transferring

    Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p
    Figure Legend Snippet: Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p

    Techniques Used:

    9) Product Images from "Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton"

    Article Title: Netrin-1 and Semaphorin 3A Promote or Inhibit Cortical Axon Branching, Respectively, by Reorganization of the Cytoskeleton

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4963-03.2004

    Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p
    Figure Legend Snippet: Netrin-1 increases F-actin, the complexity of the growth cone, and the number of filopodia and splayed microtubules along the axon shaft. A–D, Examples of cortical growth cones fixed and double labeled for F-actin (phalloidin stain) MTs (anti-tyrosinated microtubule label) after 0–60 min of netrin-1 addition (250 ng/ml). E, Bar graphs showing the increase in F-actin intensity after addition of netrin-1. For all graphs, * p

    Techniques Used: Labeling, Staining

    Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.
    Figure Legend Snippet: Receptors for Sema3A and netrin-1 are distributed along axons of cortical neurons. A , Two examples of P0 cortical neurons labeled with antibodies to the netrin-1 receptor DCC. Note the even punctate distribution of DCC along the axon shaft, in newly formed branches (top panel), and in growth cones (bottom panel). B, An individual E14 cortical neuron labeled with antibodies to neuropilin-1 and plexin-A1. Note the even distribution of the Sema3A receptor neuropilin-1 and the distal concentration of the Sema3A receptor plexin-A1 on the axon and growth cone. Scale bar, 10 μm.

    Techniques Used: Labeling, Droplet Countercurrent Chromatography, Concentration Assay

    Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.
    Figure Legend Snippet: Netrin-1 induces axon branching from filopodial protrusions. A , Phase-contrast images of a single P1 cortical neuron observed over 12 hr in time lapse. Branches emanating from filopodial protrusions are marked with a red arrowhead; branches forming from spread lamellar regions of the axon shaft are marked with a green arrowhead. The distal tip of the primary axon is marked with a blue arrowhead in all frames. B , Composite tracings of the neuron in A overlaid on the previous time point showing the progression of branch formation and extension. The time points are color coded for clarity and correspond to the color of the tracing of the neuron. Scale bar, 50 μm.

    Techniques Used:

    Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.
    Figure Legend Snippet: Local application of netrin-1 to cortical axons induces rapid filopodial protrusions localized near the pipette tip. A , A micropipette containing 25 μg/ml netrin-1 is positioned near an axon that has branched. Within 10 min after the start of the pressure ejection of netrin-1, filopodia begin to sprout and elongate (arrow at +10 min). After 30 min of netrin-1 application, numerous filopodia have formed, and most are elongating toward the pipette tip. Note also that the growth cone has stopped elongating and begins to branch (arrow at +30 min). B, Another example of filopodial formation and elongation after application of netrin-1. In this example, filopodia (arrows) form to the left of the pipette tip but not on axon regions to the top or bottom of the image (arrowheads). Note that when the pipette is moved toward the top of the image at the +35 min time point, filopodial protrusions sprout from the more distal regions of the axon, in the vicinity of the pipette tip (arrows at +45 min). Both neurons were from P1 cortical cultures. Scale bar, 20 μm.

    Techniques Used: Transferring

    Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p
    Figure Legend Snippet: Netrin-1 and FGF-2 increase and Sema3A decreases axon branching without affecting axon outgrowth. A–C , Examples of control neurons (untreated) after 72 hr in culture. D, E, Examples of cortical neurons treated with netrin-1 (250 ng/ml) for 72 hr. F, G, Bar graphs showing that treatment (72 hr) with netrin-1 (250 ng/ml) and FGF-2 (10 ng/ml) increases axon branching and branch length, whereas Sema3A (supernatant) decreases axon branching and branch length (all treatments: ** p

    Techniques Used:

    10) Product Images from "Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm"

    Article Title: Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0145389

    The dynamic differentiation of hES cells to DE is influenced by ECMPs. ( a)  Schematic overview of the semi-optimal protocol for DE differentiation. hES cells were seeded and kept undifferentiated for 4 days to allow expansion. Subsequently the cells were treated with Wnt3a to direct the cells from the pluripotent stage into the mesendoderm stage, followed by 3 days with Activin A to direct the cells from the mesendoderm stage to the DE stage.  (b)  Representative immunofluorescence images from three independent experiments of cells cultured on the five different ECMP combinations at different time points during the DE differentiation protocol. The tested ECMP combinations were fibronectin (Fn), collagen II+fibronectin (Col2+Fn), collagen I (Col1), netrin 1+fibronectin (Ne+Fn) and vitronectin (Vn) (scale bar = 200μm).  (c)  Magnification of cultures on Col1 at day 5 (scale bar = 200μm).
    Figure Legend Snippet: The dynamic differentiation of hES cells to DE is influenced by ECMPs. ( a) Schematic overview of the semi-optimal protocol for DE differentiation. hES cells were seeded and kept undifferentiated for 4 days to allow expansion. Subsequently the cells were treated with Wnt3a to direct the cells from the pluripotent stage into the mesendoderm stage, followed by 3 days with Activin A to direct the cells from the mesendoderm stage to the DE stage. (b) Representative immunofluorescence images from three independent experiments of cells cultured on the five different ECMP combinations at different time points during the DE differentiation protocol. The tested ECMP combinations were fibronectin (Fn), collagen II+fibronectin (Col2+Fn), collagen I (Col1), netrin 1+fibronectin (Ne+Fn) and vitronectin (Vn) (scale bar = 200μm). (c) Magnification of cultures on Col1 at day 5 (scale bar = 200μm).

    Techniques Used: Immunofluorescence, Cell Culture

    11) Product Images from "Netrin-1 promotes medulloblastoma cell invasiveness and angiogenesis, and demonstrates elevated expression in tumor tissue and urine of pediatric medulloblastoma patients"

    Article Title: Netrin-1 promotes medulloblastoma cell invasiveness and angiogenesis, and demonstrates elevated expression in tumor tissue and urine of pediatric medulloblastoma patients

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-13-3116

    Netrin-1 is elevated in MB
    Figure Legend Snippet: Netrin-1 is elevated in MB

    Techniques Used:

    Measurement of urinary netrin-1 levels demonstrates clinical utility as a biomarker
    Figure Legend Snippet: Measurement of urinary netrin-1 levels demonstrates clinical utility as a biomarker

    Techniques Used: Biomarker Assay

    Inhibition of netrin-1 blocks MB cell invasion and Erk phosphorylation
    Figure Legend Snippet: Inhibition of netrin-1 blocks MB cell invasion and Erk phosphorylation

    Techniques Used: Inhibition

    Neogenin regulates netrin-1-mediated EC activation
    Figure Legend Snippet: Neogenin regulates netrin-1-mediated EC activation

    Techniques Used: Activation Assay

    The potential utility of netrin-1 as a urinary biomarker
    Figure Legend Snippet: The potential utility of netrin-1 as a urinary biomarker

    Techniques Used: Biomarker Assay

    Exogenous netrin-1 induces MB cell invasiveness
    Figure Legend Snippet: Exogenous netrin-1 induces MB cell invasiveness

    Techniques Used:

    12) Product Images from "Netrin-1 promotes medulloblastoma cell invasiveness and angiogenesis, and demonstrates elevated expression in tumor tissue and urine of pediatric medulloblastoma patients"

    Article Title: Netrin-1 promotes medulloblastoma cell invasiveness and angiogenesis, and demonstrates elevated expression in tumor tissue and urine of pediatric medulloblastoma patients

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-13-3116

    Netrin-1 is elevated in MB
    Figure Legend Snippet: Netrin-1 is elevated in MB

    Techniques Used:

    Measurement of urinary netrin-1 levels demonstrates clinical utility as a biomarker
    Figure Legend Snippet: Measurement of urinary netrin-1 levels demonstrates clinical utility as a biomarker

    Techniques Used: Biomarker Assay

    Inhibition of netrin-1 blocks MB cell invasion and Erk phosphorylation
    Figure Legend Snippet: Inhibition of netrin-1 blocks MB cell invasion and Erk phosphorylation

    Techniques Used: Inhibition

    Neogenin regulates netrin-1-mediated EC activation
    Figure Legend Snippet: Neogenin regulates netrin-1-mediated EC activation

    Techniques Used: Activation Assay

    The potential utility of netrin-1 as a urinary biomarker
    Figure Legend Snippet: The potential utility of netrin-1 as a urinary biomarker

    Techniques Used: Biomarker Assay

    Exogenous netrin-1 induces MB cell invasiveness
    Figure Legend Snippet: Exogenous netrin-1 induces MB cell invasiveness

    Techniques Used:

    13) Product Images from "Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus"

    Article Title: Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002421

    LARP1 regulates Netrin-1 microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.
    Figure Legend Snippet: LARP1 regulates Netrin-1 microsomal translation. Huh7.5 cells were transfected with control and LARP1-specific siRNAs, and infected by HCV at MOI 0.1 over 4 d. Cells were lysed and analyzed by immunoblotting for LARP1 knockdown as shown in S3 Fig . LARP1 depletion decreases the expression of Netrin-1 protein in microsomes in Huh7.5 cells. LARP1, Netrin-1, and HSP60 proteins were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments, according to the infection status of the cells through sequential extractions followed by immunoblotting. HCV core was used as an infection control.

    Techniques Used: Transfection, Infection, Expressing

    Netrin-1 mRNA and HCV NS5A are LARP1 interactants. Quantification of ribosomal protein S18 (RPS18) ( A ) and Netrin-1 ( B ) mRNA by qPCR after immunoprecipitation using LARP1 antibody (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p
    Figure Legend Snippet: Netrin-1 mRNA and HCV NS5A are LARP1 interactants. Quantification of ribosomal protein S18 (RPS18) ( A ) and Netrin-1 ( B ) mRNA by qPCR after immunoprecipitation using LARP1 antibody (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Techniques Used: Real-time Polymerase Chain Reaction, Immunoprecipitation, Standard Deviation, MANN-WHITNEY

    Netrin-1 increases HCV through the UNC5A receptor. Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p
    Figure Legend Snippet: Netrin-1 increases HCV through the UNC5A receptor. Huh7.5 cells were transfected with siRNA against each UNC5 receptor or with a nontargeting control siRNA and infected at a MOI of 0.1 24 h after seeding. Cells were then trypsinized at day five post-infection before undergoing a second siRNA transfection. Recombinant soluble Netrin-1-Fc was added to the medium 12 h after transfection. Intracellular HCV RNA was quantified by RT-qPCR at each time point (left-hand graphs; data are represented as mean ± standard deviation, n = 3, Wilcoxon test, p

    Techniques Used: Transfection, Infection, Recombinant, Quantitative RT-PCR, Standard Deviation

    Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro. A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B . Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p
    Figure Legend Snippet: Netrin-1 overexpression increases HCV RNA and specific infectivity of HCV virions in vitro whereas Netrin-1 depletion decreases HCV RNA and specific infectivity in vitro. A. Detection of VR1-HA and Netrin-1-HA in transfected Huh7.5 cells by immunoblotting with the anti-HA antibody. B . Netrin-1 overexpression enhances intracellular HCV RNA. Huh7.5 cells were transfected with the VR1-HA or the Netrin-1-HA plasmid and infected at a MOI of 0.1 the day after seeding. Intracellular HCV RNA was quantified by RT-qPCR at each time point (data are shown as mean ± standard deviation, n = 3, Wilcoxon test, p

    Techniques Used: Over Expression, Infection, In Vitro, Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

    HCV increases Netrin-1 mRNA and protein. A . Virions depletion experiment. Huh7.5 cells were infected by HCV over 3 d, and the supernatant (SN) was collected, depleted of HCV particles by ultracentrifugation, and added to naïve Huh7.5 cells. Levels of HCV RNA (left) and Netrin-1 mRNA (right) were quantified by RT-qPCR. Data are represented as mean ± standard deviation ( n = 3). B . HCV increases the association of Netrin-1 mRNA with microsomes (endoplasmic reticulum [ER] membrane)-bound polysomes in Huh7.5 cells. Netrin-1 , Glucuronidase ( GUS) , and phosphomannomutase 1 ( PMM1) mRNA were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments according to the infection status of the cells through sequential extractions followed by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p
    Figure Legend Snippet: HCV increases Netrin-1 mRNA and protein. A . Virions depletion experiment. Huh7.5 cells were infected by HCV over 3 d, and the supernatant (SN) was collected, depleted of HCV particles by ultracentrifugation, and added to naïve Huh7.5 cells. Levels of HCV RNA (left) and Netrin-1 mRNA (right) were quantified by RT-qPCR. Data are represented as mean ± standard deviation ( n = 3). B . HCV increases the association of Netrin-1 mRNA with microsomes (endoplasmic reticulum [ER] membrane)-bound polysomes in Huh7.5 cells. Netrin-1 , Glucuronidase ( GUS) , and phosphomannomutase 1 ( PMM1) mRNA were monitored for their partitioning in the cytosolic (free polysomes) and microsome (ER membrane-bound polysomes) compartments according to the infection status of the cells through sequential extractions followed by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Techniques Used: Infection, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY

    HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients. A . HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D. Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients ( C,D left panels ) and nonresponding patients (C,D, right panels ). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples ( n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p
    Figure Legend Snippet: HCV levels correlate with the expression of Netrin-1 in the liver biopsies of HCV-infected patients. A . HCV-positive samples exhibit the highest levels of Netrin-1 mRNA of all the chronic liver disease biopsies. The levels of Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test. B. Positive correlation between intrahepatic levels of HCV and Netrin-1 mRNA. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR. Statistical significance was determined using the Spearman test. An outlier test was run to confirm these results. C and D. Netrin-1 mRNA parallels HCV RNA levels upon treatment. HCV RNA and Netrin-1 mRNA were quantified by RT-qPCR in paired biopsies, before and after treatment, of partially responding patients ( C,D left panels ) and nonresponding patients (C,D, right panels ). E. Parenchymal Netrin-1 staining is associated with HCV infection status in HCV-infected patients. Uninfected, chronic liver disease samples (non-cirrhotic sample, n = 1; alcohol-related cirrhosis samples, n = 3) and HCV genotype 1-infected cirrhosis samples ( n = 4) were analyzed. Representative images of Netrin-1 staining (upper panels) and HCV E2 staining (lower panels) are shown. F. Netrin-1 protein expression is increased in HCV-positive samples. The level of Netrin-1 was quantified by immunoblotting using recombinant Netrin-1 (rec. Net) as a control. G. The levels of Netrin-1 mRNA are higher in HCV+ versus HCV- biopsies, regardless of the histological stage, from normal liver to HCC. Intrahepatic Netrin-1 mRNA levels were quantified by RT-qPCR. Statistical significance was determined using the Mann-Whitney test, p

    Techniques Used: Expressing, Infection, Quantitative RT-PCR, MANN-WHITNEY, Staining, Recombinant

    HCV induces the expression of Netrin-1 in vitro. A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding ( n = 4 independent preparations from four different patients, Wilcoxon test, p
    Figure Legend Snippet: HCV induces the expression of Netrin-1 in vitro. A, B, C, D. HCV induces Netrin-1 mRNA in primary human hepatocytes. Cells were infected at a MOI of 1 with HCV genotype 2 strain 4 d after seeding ( n = 4 independent preparations from four different patients, Wilcoxon test, p

    Techniques Used: Expressing, In Vitro, Infection

    Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p
    Figure Legend Snippet: Netrin-1 enhances HCVpp entry. RNAi-mediated knockdown of EGFR. Huh7.5 cells were transfected with anti-EGFR siRNAs #3 and #4 of a previously described study [ 3 ] and infected at a MOI of 0.1. Cells were collected 3 d post-infection and analyzed by RT-qPCR ( A ) and immunoblotting using an anti-EGFR antibody targeting an extracellular epitope of the protein ( B ) (data are represented as mean ± standard deviation, n = 4, Mann-Whitney test, p

    Techniques Used: Transfection, Infection, Quantitative RT-PCR, Standard Deviation, MANN-WHITNEY

    Netrin-1 impedes EGFR recycling. Huh7.5 cells infected with HCV (day four p.i.) were transfected with siRNA (control or Netrin-1-specific) or with plasmids (VR1- or Netrin-1-expressing), serum-starved for 16 h, and incubated with EGF for 5 or 15 min prior to fixation. Cells were subsequently stained for EEA1 and EGFR and visualized by confocal microscopy. A . Li diagrams and Li coefficient calculations ( B ) for Netrin-1 knockdown and forced expression experiments. Pixels present on the left and right sides of the y -axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the imageJ software ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given experiment ( n = 2). C . Representative immunofluorescence-based localization of EEA1 and EGFR. Nuclei were counterstained with Hoechst 33342. EEA1 (green) and EGFR (red) were detected using Alexa-488 and Alexa-594, respectively. Overlays were generated by the Leica LAS AF software upon image acquisition. Open and solid arrows show partial and total colocalization, respectively. Bar = 5 μm. D . Statistical assessment of the colocalization of EEA1 and EGFR. Red (EGFR) and green (EEA1) fluorescence intensities were measured for each pixel along a 5 μm horizontal line centered around the arrow tips in ( C ) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data .
    Figure Legend Snippet: Netrin-1 impedes EGFR recycling. Huh7.5 cells infected with HCV (day four p.i.) were transfected with siRNA (control or Netrin-1-specific) or with plasmids (VR1- or Netrin-1-expressing), serum-starved for 16 h, and incubated with EGF for 5 or 15 min prior to fixation. Cells were subsequently stained for EEA1 and EGFR and visualized by confocal microscopy. A . Li diagrams and Li coefficient calculations ( B ) for Netrin-1 knockdown and forced expression experiments. Pixels present on the left and right sides of the y -axis, i.e., associated with negative and positive staining amplitude values, indicate exclusion and colocalization, respectively. Li coefficients were calculated using the JACop plugin from the imageJ software ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ). Twelve to 15 random fields were acquired per biological sample, totaling 250–300 cells analyzed for each biological sample in a given experiment ( n = 2). C . Representative immunofluorescence-based localization of EEA1 and EGFR. Nuclei were counterstained with Hoechst 33342. EEA1 (green) and EGFR (red) were detected using Alexa-488 and Alexa-594, respectively. Overlays were generated by the Leica LAS AF software upon image acquisition. Open and solid arrows show partial and total colocalization, respectively. Bar = 5 μm. D . Statistical assessment of the colocalization of EEA1 and EGFR. Red (EGFR) and green (EEA1) fluorescence intensities were measured for each pixel along a 5 μm horizontal line centered around the arrow tips in ( C ) using the Plot Profile function of the ImageJ software. Spearman correlation coefficients for each couple of intensity values are shown. The underlying data for panels in this figure can be found in S1 Data .

    Techniques Used: Infection, Transfection, Expressing, Incubation, Staining, Confocal Microscopy, Software, Immunofluorescence, Generated, Fluorescence

    Netrin-1 is virion-bound and participates in the infectivity of viral particles. HCV particles produced in cells overexpressing Netrin-1 have been preincubated with Netrin-1 antagonists and TCID 50 has been performed. A . Method validation: inhibition of HCV infectivity after incubation with anti-E2 antibody. The anti-E2 antibody was used as a control of neutralization of infectivity. B . Inhibition of HCV infectivity after incubation with three distinct Netrin-1 antagonists (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p
    Figure Legend Snippet: Netrin-1 is virion-bound and participates in the infectivity of viral particles. HCV particles produced in cells overexpressing Netrin-1 have been preincubated with Netrin-1 antagonists and TCID 50 has been performed. A . Method validation: inhibition of HCV infectivity after incubation with anti-E2 antibody. The anti-E2 antibody was used as a control of neutralization of infectivity. B . Inhibition of HCV infectivity after incubation with three distinct Netrin-1 antagonists (data are represented as mean ± standard deviation, n = 3, Mann-Whitney test, p

    Techniques Used: Infection, Produced, Inhibition, Incubation, Neutralization, Standard Deviation, MANN-WHITNEY

    14) Product Images from "Netrin-1-induced Local ?-actin Synthesis and Growth Cone Guidance Requires Zipcode Binding Protein 1"

    Article Title: Netrin-1-induced Local ?-actin Synthesis and Growth Cone Guidance Requires Zipcode Binding Protein 1

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.0166-11.2011

    ZBP1 is required for netrin-1 stimulated local translation of a β-actin reporter in growth cones (A and B) Neurons from either Zbp1 +/+ (A) or Zbp1 −/− (B) embryos were transduced with PalX2-Dendra2-β-actin 3’UTR. Growth cones can be seen prior to conversion in “DIC” (left most panel), followed by “Pre Conversion” (overlay of green and red channels, but green fluorescence predominates), and also “Post Conversion” (overlay of green and red channels, but after UV conversion red fluorescence predominates). Following netrin-1 addition, the green fluorescence increases after 30 minutes in Zbp1 +/+ growth cones, but not those from Zbp1 −/− embryos (last two images in the series are the green channel only and shown in false color; See figure legend 6A for explanation of false color scale.). (C) Application of netrin-1 results in an increase in green fluorescence in Zbp1 +/+ growth cones transduced with PalX2-Dendra2-β-actin 3’UTR, as compared to Zbp1 −/− growth cones transduced with the same construct. Neurons that received a vehicle control instead of netrin-1 or were transduced with a construct without the β-actin 3’UTR did not show an increase in fluorescence over basal translation rates. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.01, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.01). (D) Data shown in C, but only at the 30 minute time point. (E) Q-FISH to dendra2 mRNA demonstrates that the average intensity of dendra2 mRNA in Zbp1 −/− growth cones was significantly reduced as compared to wild-type neurons. Both neuronal types were transduced with the PalX2-Dendra2-β-actin 3’UTR construct. *p ≤ 0.05, Mann-Whitney. (F) Preincubation with a protein synthesis inhibitor, anisomycin, prior to stimulation with netrin-1 abolishes the increase in β-actin reporter translation. All groups were treated with netrin-1 in this experiment and “+Vehicle” in the figure legend refers to the vehicle for anisomycin. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.025, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.025).
    Figure Legend Snippet: ZBP1 is required for netrin-1 stimulated local translation of a β-actin reporter in growth cones (A and B) Neurons from either Zbp1 +/+ (A) or Zbp1 −/− (B) embryos were transduced with PalX2-Dendra2-β-actin 3’UTR. Growth cones can be seen prior to conversion in “DIC” (left most panel), followed by “Pre Conversion” (overlay of green and red channels, but green fluorescence predominates), and also “Post Conversion” (overlay of green and red channels, but after UV conversion red fluorescence predominates). Following netrin-1 addition, the green fluorescence increases after 30 minutes in Zbp1 +/+ growth cones, but not those from Zbp1 −/− embryos (last two images in the series are the green channel only and shown in false color; See figure legend 6A for explanation of false color scale.). (C) Application of netrin-1 results in an increase in green fluorescence in Zbp1 +/+ growth cones transduced with PalX2-Dendra2-β-actin 3’UTR, as compared to Zbp1 −/− growth cones transduced with the same construct. Neurons that received a vehicle control instead of netrin-1 or were transduced with a construct without the β-actin 3’UTR did not show an increase in fluorescence over basal translation rates. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.01, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.01). (D) Data shown in C, but only at the 30 minute time point. (E) Q-FISH to dendra2 mRNA demonstrates that the average intensity of dendra2 mRNA in Zbp1 −/− growth cones was significantly reduced as compared to wild-type neurons. Both neuronal types were transduced with the PalX2-Dendra2-β-actin 3’UTR construct. *p ≤ 0.05, Mann-Whitney. (F) Preincubation with a protein synthesis inhibitor, anisomycin, prior to stimulation with netrin-1 abolishes the increase in β-actin reporter translation. All groups were treated with netrin-1 in this experiment and “+Vehicle” in the figure legend refers to the vehicle for anisomycin. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.025, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.025).

    Techniques Used: Transduction, Fluorescence, Construct, Fluorescence In Situ Hybridization, MANN-WHITNEY

    Rat cortical neurons demonstrate a protein synthesis dependent attractive turning response to either netrin-1 or BDNF (A and B) Images of axonal growth cones, cultured from rat cortical neuron balls, before and after 30 minutes of exposure to a netrin-1 gradient (A, 0min; B, 30min). The tip of the micropipette can be seen in the upper left hand corner of the image. Axons subjected to turning assays were over 1mm in length (see Materials and Methods). Scale bar, 20µm. (C) Growth cones exposed to netrin-1 or BDNF exhibit positive turning angles, as compared to vehicle control. *p ≤ 0.017, Kruskal-Wallis and Mann-Whitney post-hoc with Bonferroni correction (alpha = 0.017). (D) Cumulative distribution plot of turning angles representing the individual growth cones in C. (E) The outgrowth rate over the time course of the experiment was not significantly different between the groups. One-way ANOVA. (F and I) Preincubation with a protein synthesis inhibitor, anisomycin (Aniso, 40 µM), abolishes the attractive turning response of growth cones to netrin-1 (F) and BDNF (I). *p ≤ 0.05, Mann-Whitney. (G and J) Cumulative distribution plot of turning angles representing the individual growth cones in F and I. (H and K) The outgrowth rate over the time course of the experiment was not significantly different between the groups. Student’s t-test.
    Figure Legend Snippet: Rat cortical neurons demonstrate a protein synthesis dependent attractive turning response to either netrin-1 or BDNF (A and B) Images of axonal growth cones, cultured from rat cortical neuron balls, before and after 30 minutes of exposure to a netrin-1 gradient (A, 0min; B, 30min). The tip of the micropipette can be seen in the upper left hand corner of the image. Axons subjected to turning assays were over 1mm in length (see Materials and Methods). Scale bar, 20µm. (C) Growth cones exposed to netrin-1 or BDNF exhibit positive turning angles, as compared to vehicle control. *p ≤ 0.017, Kruskal-Wallis and Mann-Whitney post-hoc with Bonferroni correction (alpha = 0.017). (D) Cumulative distribution plot of turning angles representing the individual growth cones in C. (E) The outgrowth rate over the time course of the experiment was not significantly different between the groups. One-way ANOVA. (F and I) Preincubation with a protein synthesis inhibitor, anisomycin (Aniso, 40 µM), abolishes the attractive turning response of growth cones to netrin-1 (F) and BDNF (I). *p ≤ 0.05, Mann-Whitney. (G and J) Cumulative distribution plot of turning angles representing the individual growth cones in F and I. (H and K) The outgrowth rate over the time course of the experiment was not significantly different between the groups. Student’s t-test.

    Techniques Used: Cell Culture, MANN-WHITNEY

    ZBP1 is required for netrin-1 and BDNF-induced attractive turning (A and D) Zbp1 −/− neurons do not show the attractive turning response to netrin-1 (A) and BDNF (D) exhibited by wild-type neurons. *p ≤ 0.05, Mann-Whitney. (B and E) Cumulative distribution plot of turning angles representing the individual growth cones in A and D. (C and F) The outgrowth rate over the time course of the experiments was not significantly different between the groups. *p ≤ 0.05, Mann-Whitney. (G–I) Overexpression of ZBP1-Y396F in rat cortical neurons results in the loss of netrin-1 induced attractive turning demonstrated by those neurons overexpressing ZBP1-wt (G and H), however, the outgrowth rate is not significantly different between the two groups (I). *p ≤ 0.05, Mann-Whitney.
    Figure Legend Snippet: ZBP1 is required for netrin-1 and BDNF-induced attractive turning (A and D) Zbp1 −/− neurons do not show the attractive turning response to netrin-1 (A) and BDNF (D) exhibited by wild-type neurons. *p ≤ 0.05, Mann-Whitney. (B and E) Cumulative distribution plot of turning angles representing the individual growth cones in A and D. (C and F) The outgrowth rate over the time course of the experiments was not significantly different between the groups. *p ≤ 0.05, Mann-Whitney. (G–I) Overexpression of ZBP1-Y396F in rat cortical neurons results in the loss of netrin-1 induced attractive turning demonstrated by those neurons overexpressing ZBP1-wt (G and H), however, the outgrowth rate is not significantly different between the two groups (I). *p ≤ 0.05, Mann-Whitney.

    Techniques Used: MANN-WHITNEY, Over Expression

    15) Product Images from "Netrin-1-induced Local ?-actin Synthesis and Growth Cone Guidance Requires Zipcode Binding Protein 1"

    Article Title: Netrin-1-induced Local ?-actin Synthesis and Growth Cone Guidance Requires Zipcode Binding Protein 1

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.0166-11.2011

    ZBP1 is required for netrin-1 stimulated local translation of a β-actin reporter in growth cones (A and B) Neurons from either Zbp1 +/+ (A) or Zbp1 −/− (B) embryos were transduced with PalX2-Dendra2-β-actin 3’UTR. Growth cones can be seen prior to conversion in “DIC” (left most panel), followed by “Pre Conversion” (overlay of green and red channels, but green fluorescence predominates), and also “Post Conversion” (overlay of green and red channels, but after UV conversion red fluorescence predominates). Following netrin-1 addition, the green fluorescence increases after 30 minutes in Zbp1 +/+ growth cones, but not those from Zbp1 −/− embryos (last two images in the series are the green channel only and shown in false color; See figure legend 6A for explanation of false color scale.). (C) Application of netrin-1 results in an increase in green fluorescence in Zbp1 +/+ growth cones transduced with PalX2-Dendra2-β-actin 3’UTR, as compared to Zbp1 −/− growth cones transduced with the same construct. Neurons that received a vehicle control instead of netrin-1 or were transduced with a construct without the β-actin 3’UTR did not show an increase in fluorescence over basal translation rates. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.01, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.01). (D) Data shown in C, but only at the 30 minute time point. (E) Q-FISH to dendra2 mRNA demonstrates that the average intensity of dendra2 mRNA in Zbp1 −/− growth cones was significantly reduced as compared to wild-type neurons. Both neuronal types were transduced with the PalX2-Dendra2-β-actin 3’UTR construct. *p ≤ 0.05, Mann-Whitney. (F) Preincubation with a protein synthesis inhibitor, anisomycin, prior to stimulation with netrin-1 abolishes the increase in β-actin reporter translation. All groups were treated with netrin-1 in this experiment and “+Vehicle” in the figure legend refers to the vehicle for anisomycin. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.025, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.025).
    Figure Legend Snippet: ZBP1 is required for netrin-1 stimulated local translation of a β-actin reporter in growth cones (A and B) Neurons from either Zbp1 +/+ (A) or Zbp1 −/− (B) embryos were transduced with PalX2-Dendra2-β-actin 3’UTR. Growth cones can be seen prior to conversion in “DIC” (left most panel), followed by “Pre Conversion” (overlay of green and red channels, but green fluorescence predominates), and also “Post Conversion” (overlay of green and red channels, but after UV conversion red fluorescence predominates). Following netrin-1 addition, the green fluorescence increases after 30 minutes in Zbp1 +/+ growth cones, but not those from Zbp1 −/− embryos (last two images in the series are the green channel only and shown in false color; See figure legend 6A for explanation of false color scale.). (C) Application of netrin-1 results in an increase in green fluorescence in Zbp1 +/+ growth cones transduced with PalX2-Dendra2-β-actin 3’UTR, as compared to Zbp1 −/− growth cones transduced with the same construct. Neurons that received a vehicle control instead of netrin-1 or were transduced with a construct without the β-actin 3’UTR did not show an increase in fluorescence over basal translation rates. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.01, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.01). (D) Data shown in C, but only at the 30 minute time point. (E) Q-FISH to dendra2 mRNA demonstrates that the average intensity of dendra2 mRNA in Zbp1 −/− growth cones was significantly reduced as compared to wild-type neurons. Both neuronal types were transduced with the PalX2-Dendra2-β-actin 3’UTR construct. *p ≤ 0.05, Mann-Whitney. (F) Preincubation with a protein synthesis inhibitor, anisomycin, prior to stimulation with netrin-1 abolishes the increase in β-actin reporter translation. All groups were treated with netrin-1 in this experiment and “+Vehicle” in the figure legend refers to the vehicle for anisomycin. Data is represented as ΔF/F 0 and then multiplied by 100. *p ≤ 0.025, Repeated measures ANOVA with Bonferroni post-hoc and correction (alpha=0.025).

    Techniques Used: Transduction, Fluorescence, Construct, Fluorescence In Situ Hybridization, MANN-WHITNEY

    Rat cortical neurons demonstrate a protein synthesis dependent attractive turning response to either netrin-1 or BDNF (A and B) Images of axonal growth cones, cultured from rat cortical neuron balls, before and after 30 minutes of exposure to a netrin-1 gradient (A, 0min; B, 30min). The tip of the micropipette can be seen in the upper left hand corner of the image. Axons subjected to turning assays were over 1mm in length (see Materials and Methods). Scale bar, 20µm. (C) Growth cones exposed to netrin-1 or BDNF exhibit positive turning angles, as compared to vehicle control. *p ≤ 0.017, Kruskal-Wallis and Mann-Whitney post-hoc with Bonferroni correction (alpha = 0.017). (D) Cumulative distribution plot of turning angles representing the individual growth cones in C. (E) The outgrowth rate over the time course of the experiment was not significantly different between the groups. One-way ANOVA. (F and I) Preincubation with a protein synthesis inhibitor, anisomycin (Aniso, 40 µM), abolishes the attractive turning response of growth cones to netrin-1 (F) and BDNF (I). *p ≤ 0.05, Mann-Whitney. (G and J) Cumulative distribution plot of turning angles representing the individual growth cones in F and I. (H and K) The outgrowth rate over the time course of the experiment was not significantly different between the groups. Student’s t-test.
    Figure Legend Snippet: Rat cortical neurons demonstrate a protein synthesis dependent attractive turning response to either netrin-1 or BDNF (A and B) Images of axonal growth cones, cultured from rat cortical neuron balls, before and after 30 minutes of exposure to a netrin-1 gradient (A, 0min; B, 30min). The tip of the micropipette can be seen in the upper left hand corner of the image. Axons subjected to turning assays were over 1mm in length (see Materials and Methods). Scale bar, 20µm. (C) Growth cones exposed to netrin-1 or BDNF exhibit positive turning angles, as compared to vehicle control. *p ≤ 0.017, Kruskal-Wallis and Mann-Whitney post-hoc with Bonferroni correction (alpha = 0.017). (D) Cumulative distribution plot of turning angles representing the individual growth cones in C. (E) The outgrowth rate over the time course of the experiment was not significantly different between the groups. One-way ANOVA. (F and I) Preincubation with a protein synthesis inhibitor, anisomycin (Aniso, 40 µM), abolishes the attractive turning response of growth cones to netrin-1 (F) and BDNF (I). *p ≤ 0.05, Mann-Whitney. (G and J) Cumulative distribution plot of turning angles representing the individual growth cones in F and I. (H and K) The outgrowth rate over the time course of the experiment was not significantly different between the groups. Student’s t-test.

    Techniques Used: Cell Culture, MANN-WHITNEY

    ZBP1 is required for netrin-1 and BDNF-induced attractive turning (A and D) Zbp1 −/− neurons do not show the attractive turning response to netrin-1 (A) and BDNF (D) exhibited by wild-type neurons. *p ≤ 0.05, Mann-Whitney. (B and E) Cumulative distribution plot of turning angles representing the individual growth cones in A and D. (C and F) The outgrowth rate over the time course of the experiments was not significantly different between the groups. *p ≤ 0.05, Mann-Whitney. (G–I) Overexpression of ZBP1-Y396F in rat cortical neurons results in the loss of netrin-1 induced attractive turning demonstrated by those neurons overexpressing ZBP1-wt (G and H), however, the outgrowth rate is not significantly different between the two groups (I). *p ≤ 0.05, Mann-Whitney.
    Figure Legend Snippet: ZBP1 is required for netrin-1 and BDNF-induced attractive turning (A and D) Zbp1 −/− neurons do not show the attractive turning response to netrin-1 (A) and BDNF (D) exhibited by wild-type neurons. *p ≤ 0.05, Mann-Whitney. (B and E) Cumulative distribution plot of turning angles representing the individual growth cones in A and D. (C and F) The outgrowth rate over the time course of the experiments was not significantly different between the groups. *p ≤ 0.05, Mann-Whitney. (G–I) Overexpression of ZBP1-Y396F in rat cortical neurons results in the loss of netrin-1 induced attractive turning demonstrated by those neurons overexpressing ZBP1-wt (G and H), however, the outgrowth rate is not significantly different between the two groups (I). *p ≤ 0.05, Mann-Whitney.

    Techniques Used: MANN-WHITNEY, Over Expression

    16) Product Images from "Cue-Polarized Transport of β-actin mRNA Depends on 3′UTR and Microtubules in Live Growth Cones"

    Article Title: Cue-Polarized Transport of β-actin mRNA Depends on 3′UTR and Microtubules in Live Growth Cones

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00300

    Netrin-1 induced RNA movement to growth cone periphery depends on dynamic microtubules and not F-actin. (A) Fluorescence micrographs show the relative distributions of F-actin (phalloidin staining) and dynamic microtubules (tyryosinated-tubulin) in distal axon. (B–C) Dynamic microtubule and F-actin were selectively disrupted to test which cytoskeletal element was required for facilitating netrin-1 induced peripheral mRNA localization. Unlike with intact cytoskeleton ( B , Netrin-1 only; n = 17 growth cones), disrupting dynamic microtubules with colchicine ( C , Colchicine + Netrin-1; n = 4 growth cones) abolished the netrin-1-induced increase in peripheral mRNA localization. In the presence of cytochalasin D ( C , Cytochalasin D + Netrin-1; n = 9 growth cones), which disrupts F-actin, mRNA granules still exhibited increased peripheral localization in response to netrin-1 stimulation. Each panel in (B,C) shows the same growth cone before and after netrin-1 treatment. (D) Quantification of the number of peripherally localized mRNA granules. Dotted lines represent least-squares fits to a Lorentzian function. (Netrin vs. Netrin + Cytochalasin D: F (3, 243) = 2.405, p = 0.068; Netrin vs. Netrin + Colchicine: F (3, 222) = 25.75, *** p
    Figure Legend Snippet: Netrin-1 induced RNA movement to growth cone periphery depends on dynamic microtubules and not F-actin. (A) Fluorescence micrographs show the relative distributions of F-actin (phalloidin staining) and dynamic microtubules (tyryosinated-tubulin) in distal axon. (B–C) Dynamic microtubule and F-actin were selectively disrupted to test which cytoskeletal element was required for facilitating netrin-1 induced peripheral mRNA localization. Unlike with intact cytoskeleton ( B , Netrin-1 only; n = 17 growth cones), disrupting dynamic microtubules with colchicine ( C , Colchicine + Netrin-1; n = 4 growth cones) abolished the netrin-1-induced increase in peripheral mRNA localization. In the presence of cytochalasin D ( C , Cytochalasin D + Netrin-1; n = 9 growth cones), which disrupts F-actin, mRNA granules still exhibited increased peripheral localization in response to netrin-1 stimulation. Each panel in (B,C) shows the same growth cone before and after netrin-1 treatment. (D) Quantification of the number of peripherally localized mRNA granules. Dotted lines represent least-squares fits to a Lorentzian function. (Netrin vs. Netrin + Cytochalasin D: F (3, 243) = 2.405, p = 0.068; Netrin vs. Netrin + Colchicine: F (3, 222) = 25.75, *** p

    Techniques Used: Fluorescence, Staining

    Global netrin-1 stimulation changes the dynamics of β-actin mRNA granules. Axons were imaged at 1 frame per second from 1 min before bath application of netrin-1, and the imaging continued for up to 10 min after netrin application. (A) The numbers of β-actin mRNA granules passing three chosen locations (dashed lines) along the axon shaft were scored. The number of anterograde-moving granules increased upon netrin-1 treatment, reaching peak value at 5–6 min and returning to the base line by 8 min of netrin-1 treatment. The number of retrograde-moving granules showed moderate decrease with time upon netrin-1 treatment. ( n = 9 axons) Anterograde vs. retrograde [ F (7, 120) = 3.775, ### p = 0.001]; Vs. T = −1 min, ** p
    Figure Legend Snippet: Global netrin-1 stimulation changes the dynamics of β-actin mRNA granules. Axons were imaged at 1 frame per second from 1 min before bath application of netrin-1, and the imaging continued for up to 10 min after netrin application. (A) The numbers of β-actin mRNA granules passing three chosen locations (dashed lines) along the axon shaft were scored. The number of anterograde-moving granules increased upon netrin-1 treatment, reaching peak value at 5–6 min and returning to the base line by 8 min of netrin-1 treatment. The number of retrograde-moving granules showed moderate decrease with time upon netrin-1 treatment. ( n = 9 axons) Anterograde vs. retrograde [ F (7, 120) = 3.775, ### p = 0.001]; Vs. T = −1 min, ** p

    Techniques Used: Imaging

    Netrin-1 gradient induces asymmetric localization of β-actin mRNA in growth cone. (A) Growth cones were imaged at 1 frame per second in the presence a netrin-1 gradient set up from a micropipette perpendicular to the growth cone. (B) The mean x-coordinate of all β-actin mRNA granules at each timepoint was compared to the mean x-coordinate at time 0. With the source of netrin-1 on the left, a negative shift value indicates a shift of β -actin mRNA granules toward the netrin-1 source. (C) Relative granule centroid shift values are plotted. ( n = 15 growth cones for full length 3′UTR at 1fps for 5–15 min; n = 9 growth cones for Δ3′UTR at 1fps for 5–15 min) Black dotted lines represent least-square fits to a linear function. [ F (1, 407) = 98.27, *** p
    Figure Legend Snippet: Netrin-1 gradient induces asymmetric localization of β-actin mRNA in growth cone. (A) Growth cones were imaged at 1 frame per second in the presence a netrin-1 gradient set up from a micropipette perpendicular to the growth cone. (B) The mean x-coordinate of all β-actin mRNA granules at each timepoint was compared to the mean x-coordinate at time 0. With the source of netrin-1 on the left, a negative shift value indicates a shift of β -actin mRNA granules toward the netrin-1 source. (C) Relative granule centroid shift values are plotted. ( n = 15 growth cones for full length 3′UTR at 1fps for 5–15 min; n = 9 growth cones for Δ3′UTR at 1fps for 5–15 min) Black dotted lines represent least-square fits to a linear function. [ F (1, 407) = 98.27, *** p

    Techniques Used:

    17) Product Images from "RNA binding protein Vg1RBP regulates terminal arbor formation but not long-range axon navigation in the developing visual system"

    Article Title: RNA binding protein Vg1RBP regulates terminal arbor formation but not long-range axon navigation in the developing visual system

    Journal: Developmental neurobiology

    doi: 10.1002/dneu.22110

    ΔKH4-eGFP blocks attractive growth cone turning in response to netrin-1 in vitro . A-D, Phase contrast images of control (A, B) and ΔKH4-eGFP-expressing (C, D) growth cones at the start and 60 min after, exposure to a diffusible gradient of netrin-1. The position of the pipette ejecting netrin-1 is visible in the top right corner of each image. Scale bar, 20 μm. E-G, Traces of the trajectory of growth cones during 60 min exposure to a netrin-1 gradient. The origin represents the position of the growth at the start of the experiment. Axes represent μm. Netrin-1 stimulated a significantly different mean turning angle in control (15.47° ± 5.68°; P = 0.0159, t test) but not ΔKH4-eGFP-expressing growth cones (−18.40° ± 4.77°; P = 0.117), compared with the mean turning angle of control growth cones stimulated with vehicle solution (−5.75° ± 5.81°). H. Cumulative distribution histogram of the turning angles induced by netrin-1 and vehicle solution.
    Figure Legend Snippet: ΔKH4-eGFP blocks attractive growth cone turning in response to netrin-1 in vitro . A-D, Phase contrast images of control (A, B) and ΔKH4-eGFP-expressing (C, D) growth cones at the start and 60 min after, exposure to a diffusible gradient of netrin-1. The position of the pipette ejecting netrin-1 is visible in the top right corner of each image. Scale bar, 20 μm. E-G, Traces of the trajectory of growth cones during 60 min exposure to a netrin-1 gradient. The origin represents the position of the growth at the start of the experiment. Axes represent μm. Netrin-1 stimulated a significantly different mean turning angle in control (15.47° ± 5.68°; P = 0.0159, t test) but not ΔKH4-eGFP-expressing growth cones (−18.40° ± 4.77°; P = 0.117), compared with the mean turning angle of control growth cones stimulated with vehicle solution (−5.75° ± 5.81°). H. Cumulative distribution histogram of the turning angles induced by netrin-1 and vehicle solution.

    Techniques Used: In Vitro, Expressing, Transferring

    18) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    19) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    20) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    21) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    22) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    23) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    24) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    25) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    26) Product Images from "Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics"

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00298

    Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p
    Figure Legend Snippet: Netrin-1 modulates cyclic nucleotides differentially for the local and global treatments. (A) Pseudo-colored cAMP CFP:YFP fluorescence intensity ratio (ΔR), normalized by the baseline ratio (R), in distal axons in response to axonal treatment with Netrin-1 or vehicle (control). Scale bars = 5 μm. (B) cAMP and cGMP signals in growth cones and in cell bodies in response to treatments (arrows) with vehicle (gray) or with local or global Netrin-1 treatments (black). Growth cone cAMP signals are given separately for responsive (red) and unresponsive (blue) axons. Data are given as mean with 95% confidence interval (broken lines). * p

    Techniques Used: Fluorescence

    Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.
    Figure Legend Snippet: Putative mechanisms for the neuronal response to locally and globally applied Netrin-1 . Axonal and global Netrin-1 treatments differentially regulate membranous and total DCC levels, and the dynamics of second messengers. Arrows indicate the character and strength of the change as measured in our experiments (solid lines) and based on the literature (broken lines). Axonal Netrin-1 increases cAMP level, the frequency of Ca 2+ transients and the total and membranous DCC levels in growth cones. cAMP supports Ca 2+ efflux from the endoplasmic reticulum (ER); high level of Ca 2+ leads to calcium-induced calcium release (CICR). Global Netrin-1 increases total DCC levels, but not membranous DCC levels, cAMP or the frequency of calcium transients. CICR is inhibited. Axonal Netrin-1 slightly affects the axon speed, and severely decreases axon velocity (block arrows). Global Netrin-1 significantly decreases both, axon speed and velocity. Differences in axonal and global Netrin-1 responses suggest that neurons sense Netrin-1 along their entirety and alter their response accordingly; however, the mechanisms of anterograde propagation of Netrin-1-induced signals are unknown.

    Techniques Used: Droplet Countercurrent Chromatography, Blocking Assay

    Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Calcium efflux from internal stores is necessary for Netrin-1-driven DCC membrane insertion. (A) Membranous DCC (DCC memb ) staining intensity after 25 min of axonal treatments, as shown by the schematics: with vehicle (control; PBS for Netrin-1, water for Ryanodine), 100 μM ryanodine (High Ry; blue), 1.0 μg ml −1 Netrin-1 (pink), or combined (purple). The hatch shows imaging site—axonal ( A ) or somatic ( S ). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual measurements from N ≥ 3 independent experiments (Table S7 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Imaging

    Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p
    Figure Legend Snippet: Netrin-1 modulates local and long-range DCC insertion into plasma membrane. (A) Membranous DCC staining in the control group and 25 and 90 min after isolated (axonal and somatic) or global Netrin-1 treatment. Schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). The ROIs were chosen in Phalloidin channel as shown by the color overlay in control. Scale bars = 10 μm. (B) Staining intensity (mean ± s.e.m.) was measured in the axonal compartment (solid fill) and in the somatic compartment (hatch fill), and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S2 ); statistical significance compared to controls unless indicated otherwise; ** p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Isolation

    Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p
    Figure Legend Snippet: Axonal Netrin-1-induced change in membranous DCC in cell bodies is uniformly distributed within the somatic compartment. (A) Cell permeable Calcein AM labels entire neurons upon its uptake in the axonal compartment (green). Majority of neurons do not have an axon in the axonal compartment, as indicated by the nuclear stain (blue). Scale bar = 200 μm. (B) The boxed area in (A) is magnified to reveal neurons labeled and not labeled with Calcein near microchannels. Scale bar = 50 μm. (C) Membranous DCC staining intensity in cell bodies normalized by the control value for increasing duration of 1.0 μg ml −1 axonal Netrin-1 treatment. Error bars represent s.e.m.; n > 200 for each time point from N ≥ 3 distinct cultures (Table S3 ); statistical significance compared to controls with Kolmogorov-Smirnov test with Dunn-Sidak correction for multiple comparisons; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining, Labeling

    Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Netrin-1 treatment modulates local and long-range total DCC. (A) Total DCC staining upon 90 min-long somatic, axonal, or global treatment with 1.0 μg ml −1 Netrin-1 or vehicle (control). The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). Scale bars = 10 μm. (B) DCC staining intensity (mean ± s.e.m.) was measured in the ROIs in the axonal (solid fill) and somatic (hatch fill) compartments, and normalized with the control signal. n is the number of individual ROIs from N ≥ 3 independent experiments (Table S4 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Droplet Countercurrent Chromatography, Staining

    Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p
    Figure Legend Snippet: Axonal but not global Netrin-1 induces local and long-range changes in calcium activity. (A,B) Calcium activity in cell bodies before (white time overlay) and after (green time overlay) adding vehicle (A) or 1.0 μg ml −1 Netrin-1 (B) into axonal compartment at t = 0. Green arrows point at cell bodies that start firing. Broken line indicates the beginning of microchannels. Scale bar = 20 μm. (C) Representative calcium activity traces in response to treatment (pink bars) with vehicle (control) or with 1.0 μg ml −1 Netrin-1. Signals ( F ) exceeding 20% of the baseline fluorescence ( F0 ; dashed lines) were considered positive. The schematics show which compartment, somatic ( S ) or axonal ( A ), underwent Netrin-1 treatment (pink) and which compartment was imaged (hatch). (D) Frequency of calcium transients (mean ± s.e.m.); n is the number of individual measurements from N ≥ 2 distinct cultures (Table S6 ); * p

    Techniques Used: Activity Assay, Fluorescence

    Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p
    Figure Legend Snippet: Axon elongation depends on the localization of Netrin-1 treatment. (A) Neurons cultured in the bicompartmental device (inset) send neurites from the somatic ( S ) to the axonal ( A ) compartment through microchannels. Scale bar = 80 μm. (B) Neuronal growth profile at 3 and 5 days in vitro (DIV). Scale bars = 50 μm. (C) Modes of Netrin-1 treatment. The axon elongation was measured in the axonal compartment (hatch). (D) Average velocity and speed (mean ± s.e.m.) for increasing Netrin-1 concentrations and different compartments of delivery. The numbers on bars represent the number of individual axons from N ≥ 2 independent experiments (Table S1 ); statistical significance compared to controls unless indicated otherwise; * p

    Techniques Used: Cell Culture, In Vitro

    27) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    28) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    29) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    30) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    31) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    32) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    33) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    34) Product Images from "Netrin 1 regulates blood–brain barrier function and neuroinflammation"

    Article Title: Netrin 1 regulates blood–brain barrier function and neuroinflammation

    Journal: Brain

    doi: 10.1093/brain/awv092

    Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of
    Figure Legend Snippet: Netrin 1 counteracts the detrimental outcome induced by inflammation on the human and murine blood–brain barrier . Production of IL8, MCP-1, IP-10, and IL6 by primary cultures of human brain-derived endothelial cells activated with 100 U/ml of

    Techniques Used: Derivative Assay

    Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial
    Figure Legend Snippet: Netrin 1 expression at the blood–brain barrier . Quantitative RNA sequencing analysis ( A ), quantitative PCR ( B ) and western blot ( C ) analyses of netrin 1 (N1) expression by primary cultures of human brain-derived endothelial cells. Endothelial

    Techniques Used: Expressing, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Western Blot, Derivative Assay

    Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally
    Figure Legend Snippet: Netrin 1 reduces severity of EAE . Mean cumulative clinical scores ( A and E ), score prevalence per treatment group ( B and F ), disease incidence ( C and G ) and weight change ( D and H ) were recorded from MOG 35-55 -immunized C57BL/6 mice injected intraperitoneally

    Techniques Used: Mouse Assay, Injection

    Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte
    Figure Legend Snippet: Netrin 1 regulates junctional protein levels in vitro and in vivo . Expression of junctional proteins JAM-A ( A ), occludin ( B ), and claudin-5 ( C ) in primary cultures of human brain-derived endothelial cells untreated (control, C) or treated with astrocyte

    Techniques Used: In Vitro, In Vivo, Expressing, Derivative Assay

    Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or
    Figure Legend Snippet: Netrin 1 enhances endothelial barrier properties in vitro and in vivo . Permeability coefficient of bovine serum albumin (BSA) ( A ) and dextran (D10) ( B ) across primary cultures of human brain-derived endothelial cells, either untreated/resting (R), or

    Techniques Used: In Vitro, In Vivo, Permeability, Derivative Assay

    Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic
    Figure Legend Snippet: Netrin 1 reduces plasma protein leakage across the blood–brain barrier during EAE . In situ immunostainings of immunoglobulin G (red, IgG) and fibrinogen (red) in spinal cord sections of control or netrin 1 treated EAE mice, during presymptomatic

    Techniques Used: In Situ, Mouse Assay

    35) Product Images from "Netrin-1 Ameliorates Blood-Brain Barrier Impairment Secondary to Ischemic Stroke via the Activation of PI3K Pathway"

    Article Title: Netrin-1 Ameliorates Blood-Brain Barrier Impairment Secondary to Ischemic Stroke via the Activation of PI3K Pathway

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2017.00700

    Characteristic and viability of RBMVECs. (A) RBMVECs were vWF-positive, indicating the attribute of ECs. Arrows indicates positive green signal by immunofluorescence. (B) Cell viability shown by CCK-8 and Annexin V-FITC kit. Netrin-1 (50 ng/mL) enhanced cell viability after OGD while PI3K inhibitor LY294002 prohibited the beneficial effect of netrin-1. * P
    Figure Legend Snippet: Characteristic and viability of RBMVECs. (A) RBMVECs were vWF-positive, indicating the attribute of ECs. Arrows indicates positive green signal by immunofluorescence. (B) Cell viability shown by CCK-8 and Annexin V-FITC kit. Netrin-1 (50 ng/mL) enhanced cell viability after OGD while PI3K inhibitor LY294002 prohibited the beneficial effect of netrin-1. * P

    Techniques Used: Immunofluorescence, CCK-8 Assay

    Netrin-1 receptors on RBMVECs and their function. (A) UNC5H2 receptor, not DCC receptor, was expressed on RBMVECs. Arrows indicates positive green signal by immunofluorescence. (B) Western blot analysis of UNC5H2 receptor. OGD-induced UNC5H2 was efficiently knocked down by UNC5H2 siRNA transfection. (C) Cell viability shown by CCK-8 and Annexin V-FITC kit. (D) Western blot analysis of autophagy-related protein p62 and LC3. UNC5H2 siRNA transfection did not change cell viability or autophagy activity after OGD, but suppressed the strengthened effects of netrin-1 (50 ng/mL) on cell viability and autophagy activity. * P
    Figure Legend Snippet: Netrin-1 receptors on RBMVECs and their function. (A) UNC5H2 receptor, not DCC receptor, was expressed on RBMVECs. Arrows indicates positive green signal by immunofluorescence. (B) Western blot analysis of UNC5H2 receptor. OGD-induced UNC5H2 was efficiently knocked down by UNC5H2 siRNA transfection. (C) Cell viability shown by CCK-8 and Annexin V-FITC kit. (D) Western blot analysis of autophagy-related protein p62 and LC3. UNC5H2 siRNA transfection did not change cell viability or autophagy activity after OGD, but suppressed the strengthened effects of netrin-1 (50 ng/mL) on cell viability and autophagy activity. * P

    Techniques Used: Droplet Countercurrent Chromatography, Immunofluorescence, Western Blot, Transfection, CCK-8 Assay, Activity Assay

    Western blot analysis of autophagy-related protein beclin-1, p62 and LC3. (A) Netrin-1 infusion increased autophagy activity 14 days after MCAO compared with the vehicle group, suppressed by PI3K inhibitor 3-MA. (B) Netrin-1 (50 ng/mL) enhanced autophagy activity after OGD while PI3K inhibitor LY294002 suppressed the effect of netrin-1. * P
    Figure Legend Snippet: Western blot analysis of autophagy-related protein beclin-1, p62 and LC3. (A) Netrin-1 infusion increased autophagy activity 14 days after MCAO compared with the vehicle group, suppressed by PI3K inhibitor 3-MA. (B) Netrin-1 (50 ng/mL) enhanced autophagy activity after OGD while PI3K inhibitor LY294002 suppressed the effect of netrin-1. * P

    Techniques Used: Western Blot, Activity Assay

    Neurological function assessed by mNSS. The scores were increased over different time points after MCAO, but significantly improved with netrin-1 infusion. The beneficial effect of netrin-1 was inhibited by 3-MA 14 days after MCAO. * P
    Figure Legend Snippet: Neurological function assessed by mNSS. The scores were increased over different time points after MCAO, but significantly improved with netrin-1 infusion. The beneficial effect of netrin-1 was inhibited by 3-MA 14 days after MCAO. * P

    Techniques Used:

    Secondary impairment of BBB in the ipsilateral thalamus. (A) Representative images of occludin (red) and extravasation of albumin (green), and relative amount of albumin extravasation by immunofluorescence. (B) Western blot analysis of tight junction protein ZO-1 and occludin. Extravasation of albumin and down-regulation of ZO-1 and occludin after MCAO were improved by netrin-1 infusion. PI3K inhibitor 3-MA prohibited the beneficial effect of netrin-1. * P
    Figure Legend Snippet: Secondary impairment of BBB in the ipsilateral thalamus. (A) Representative images of occludin (red) and extravasation of albumin (green), and relative amount of albumin extravasation by immunofluorescence. (B) Western blot analysis of tight junction protein ZO-1 and occludin. Extravasation of albumin and down-regulation of ZO-1 and occludin after MCAO were improved by netrin-1 infusion. PI3K inhibitor 3-MA prohibited the beneficial effect of netrin-1. * P

    Techniques Used: Immunofluorescence, Western Blot

    Detection of endothelial cells in the ipsilateral thalamus. (A) Representative images of RECA-1 (red) by immunofluorescence. (B) Western blot analysis. The positive area of RECA-1 and the protein level of VEGF were increased 14 days after MCAO. Netrin-1 infusion did not change the positive area of RECA-1 or the protein levels of vWF and VEGF after MCAO. * P
    Figure Legend Snippet: Detection of endothelial cells in the ipsilateral thalamus. (A) Representative images of RECA-1 (red) by immunofluorescence. (B) Western blot analysis. The positive area of RECA-1 and the protein level of VEGF were increased 14 days after MCAO. Netrin-1 infusion did not change the positive area of RECA-1 or the protein levels of vWF and VEGF after MCAO. * P

    Techniques Used: Immunofluorescence, Western Blot

    36) Product Images from "Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)"

    Article Title: Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0494-5

    Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p
    Figure Legend Snippet: Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p

    Techniques Used: In Vivo, Chick Chorioallantoic Membrane Assay, Derivative Assay, Incubation

    Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain
    Figure Legend Snippet: Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain

    Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Droplet Countercurrent Chromatography, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence

    RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p
    Figure Legend Snippet: RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p

    Techniques Used: In Vitro, In Vivo, Recombinant

    Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)
    Figure Legend Snippet: Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)

    Techniques Used: Derivative Assay, Expressing, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control

    Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used: In Vitro, Migration, Wound Healing Assay, Recombinant, Positive Control, Amplification

    Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used:

    Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p
    Figure Legend Snippet: Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p

    Techniques Used: Recombinant

    37) Product Images from "Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)"

    Article Title: Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0494-5

    Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p
    Figure Legend Snippet: Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p

    Techniques Used: In Vivo, Chick Chorioallantoic Membrane Assay, Derivative Assay, Incubation

    Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain
    Figure Legend Snippet: Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain

    Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Droplet Countercurrent Chromatography, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence

    RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p
    Figure Legend Snippet: RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p

    Techniques Used: In Vitro, In Vivo, Recombinant

    Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)
    Figure Legend Snippet: Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)

    Techniques Used: Derivative Assay, Expressing, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control

    Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used: In Vitro, Migration, Wound Healing Assay, Recombinant, Positive Control, Amplification

    Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used:

    Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p
    Figure Legend Snippet: Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p

    Techniques Used: Recombinant

    38) Product Images from "Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)"

    Article Title: Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0494-5

    Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p
    Figure Legend Snippet: Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p

    Techniques Used: In Vivo, Chick Chorioallantoic Membrane Assay, Derivative Assay, Incubation

    Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain
    Figure Legend Snippet: Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain

    Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Droplet Countercurrent Chromatography, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence

    RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p
    Figure Legend Snippet: RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p

    Techniques Used: In Vitro, In Vivo, Recombinant

    Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)
    Figure Legend Snippet: Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)

    Techniques Used: Derivative Assay, Expressing, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control

    Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used: In Vitro, Migration, Wound Healing Assay, Recombinant, Positive Control, Amplification

    Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used:

    Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p
    Figure Legend Snippet: Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p

    Techniques Used: Recombinant

    39) Product Images from "Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)"

    Article Title: Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0494-5

    Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p
    Figure Legend Snippet: Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p

    Techniques Used: In Vivo, Chick Chorioallantoic Membrane Assay, Derivative Assay, Incubation

    Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain
    Figure Legend Snippet: Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain

    Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Droplet Countercurrent Chromatography, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence

    RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p
    Figure Legend Snippet: RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p

    Techniques Used: In Vitro, In Vivo, Recombinant

    Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)
    Figure Legend Snippet: Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)

    Techniques Used: Derivative Assay, Expressing, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control

    Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used: In Vitro, Migration, Wound Healing Assay, Recombinant, Positive Control, Amplification

    Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used:

    Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p
    Figure Legend Snippet: Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p

    Techniques Used: Recombinant

    40) Product Images from "Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)"

    Article Title: Netrin-1 acts as a non-canonical angiogenic factor produced by human Wharton’s jelly mesenchymal stem cells (WJ-MSC)

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0494-5

    Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p
    Figure Legend Snippet: Netrin-1 contributes to angiogenesis in vivo in a CAM assay. a Representative images of distinct experimental approaches on CAM: DMEM (20 μL), Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC) (1.5*10 6 ), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 50 μM), using for each condition approximately 15 eggs (scale bar = 1 mm). b Quantification of angiogenesis after 4 days of incubation in experimental conditions as indicated. Data correspond to the mean ± S.E.M. (WJ-MSC, n = 5, * p

    Techniques Used: In Vivo, Chick Chorioallantoic Membrane Assay, Derivative Assay, Incubation

    Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain
    Figure Legend Snippet: Endothelial cells derived from human umbilical vein endothelial cells (HUVEC) express low levels of classic Netrin receptors and both Netrin-1 (NTN-1) and Netrin-4 (NTN-4) ligands. a mRNA levels of classic (■) and non-classic (□) Netrin receptors were quantified by qPCR relative to GAPDH expression ( dashed line ). Values are mean ± S.E.M. ( n = 7). b Absence of DCC expression in HUVEC was established by Western blot, while Neogenin-1 as well as UNC5b and UNC5c expression could be detected. Endogenous Netrins and vascular endothelial growth factor (VEGF) expression in HUVEC was determined by Western blot. β-actin was used as internal reference ( n = 5–7). c The non-classical Netrin receptor integrin α3β1 was also detected by flow cytometry ( n = 3). d Immunofluorescence for receptors and ligands in HUVEC (magnification × 63). eNOS (endothelial nitric oxide synthase) or CD-31 (PECAM) were used as endothelial cell markers and DAPI for nuclear counterstain

    Techniques Used: Derivative Assay, Real-time Polymerase Chain Reaction, Expressing, Droplet Countercurrent Chromatography, Western Blot, Flow Cytometry, Cytometry, Immunofluorescence

    RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p
    Figure Legend Snippet: RhoA/ROCK pathway contributes to angiogenesis in vitro and in vivo in a Netrin-1-independent manner. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM) and exoenzyme C3 transferase (Cytoskeleton, Inc., 1–1.5 μg/mL), in absence or presence of recombinant human Netrin-1 (NTN-1) (10 ng/mL). The graphs shown below represent quantified data corresponding to the mean ± S.E.M. [ n = 3, * p

    Techniques Used: In Vitro, In Vivo, Recombinant

    Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)
    Figure Legend Snippet: Netrin-1 is preferentially expressed and secreted by Wharton’s jelly-derived mesenchymal stem cells (WJ-MSC), whilst Netrin-4 expression is almost absent. Netrin-1 (NTN-1) and Netrin-4 (NTN-4) expression was confirmed by different experimental techniques. a Representative images of the histological analysis of umbilical cord sections are shown. MSCs uniformly distribute within Wharton’s jelly and show positive staining for Alcian Blue and Netrins (magnification × 20). Inset show optical zoom images of NTN-1 and NTN-4 staining ( n = 3). b Flow cytometry and immunofluorescence anti-NTN-1 and −4 confirm preferential expression of NTN-1 in WJ-MSC ( n = 3) (magnification × 63). c Western blot for NTN-1, NTN-4 and vascular endothelial growth factor (VEGF) (positive control) in whole cell lysate (CL) and conditioned media (CM) of WJ-MSC cultures. β-actin (for CL) and Ponceau (for CM) were used as internal loading controls, respectively ( n = 3–7). d ELISA analysis for NTN-1 in WJ-MSC; CM was obtained at indicated time points ( n = 4). e Western blot for NTN-1 in WJ-MSC, seeded 24 h in Integra® matrix (IM) or collagen I (Col), versus respective empty scaffold (−) used as negative control. Ligand expression was analyzed both in CL or CM. β-actin and Ponceau were used as internal loading controls, respectively ( n = 4–5)

    Techniques Used: Derivative Assay, Expressing, Staining, Flow Cytometry, Cytometry, Immunofluorescence, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay, Negative Control

    Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1 induces angiogenesis in vitro in HUVEC. a Netrin-1 (NTN-1) influence on HUVEC cell migration was determined using scratch assay, where cells were serum-starved and treated for 8 h with different recombinant human NTN-1 concentrations, as indicated. Endothelial basal media (EBM) was used as internal reference and recombinant human vascular endothelial growth factor (VEGF) (40 ng/mL) was used as a positive control. Representative pictures of each condition are shown (amplification × 10). b Quantified results correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used: In Vitro, Migration, Wound Healing Assay, Recombinant, Positive Control, Amplification

    Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p
    Figure Legend Snippet: Netrin-1, secreted by WJ-MSC, promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay treated as indicated. Cells were exposed for 4 h to DMEM, endothelial growth media (EGM), WJ-MSC-conditioned media (CM), 2F5 [a drug targeting Netrin-1 (NTN-1), 0.5 μg/μL] and CBO (a VEGF-receptor inhibitor, 20 μM), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 3, * p

    Techniques Used:

    Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p
    Figure Legend Snippet: Netrin-1 promotes angiogenesis in HUVEC. a Representative images of HUVEC tubule assay. Cells were exposed for 4 h to endothelial basal media (EBM), endothelial growth media (EGM), IgG (internal antibody control, 2 μg/mL), 2F5 [a drug targeting Netrin-1 (NTN-1), 2 μg/mL] and anti-NTN-1 antibody (R D Systems, 2 μg/mL), in absence or presence of recombinant human NTN-1 (10 ng/mL), scale bar = 15 μm. b Quantified data correspond to the mean ± S.E.M. ( n = 4, * p

    Techniques Used: Recombinant

    Related Articles

    other:

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Article Snippet: Netrin-1 is reported predominantly as an attractant for cortical neurons (Bouchard et al., ); however, several studies including ours demonstrated its repulsive effects (Powell et al., ; Blasiak et al., ).

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Article Snippet: The differential effects of Netrin-1 after axonal, somatic, and global treatments indicate that cell bodies sense Netrin-1 and contribute to the distal axonal response.

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Article Snippet: On the other hand, neurons that experience Netrin-1 globally may have altered cytoskeletal dynamics resulting in the reduction of axon speed and consequently of axon velocity.

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Article Snippet: In the cell bodies, Netrin-1 induced a very different DCCtotal response: somatic and global Netrin-1 treatments decreased DCCtotal , while axonal Netrin-1 treatment increased DCCtotal .

    Article Title: RNA binding protein Vg1RBP regulates terminal arbor formation but not long-range axon navigation in the developing visual system
    Article Snippet: One intriguing possibility is that netrin-1 can induce protein synthesis (PS)-dependent and –independent responses in growth cones, as has been found with different concentrations of Sema3A ( ; ), and that only terminal branching is PS-dependent.

    Droplet Countercurrent Chromatography:

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Article Snippet: .. Netrin-1 induces local and global changes in membranous DCC dynamics Netrin-1 modulates DCC cycling on and off the plasma membrane (Kim et al., ), which has a direct effect on axon elongation (Bouchard et al., ). .. We tested how exposing different subcellular regions to Netrin-1 affects DCC dynamics in distal axons.

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Article Snippet: .. However, considering that cortical neurons secrete Netrin-1 along their entire lengths (Bouchard et al., ), distal axons may be exposed to sufficiently high levels of endogenous Netrin-1 that occupies their membranous DCC to prevent apoptotic induction. .. Our results suggest that neurons sense Netrin-1 along their entire lengths and adjust their DCCmemb accordingly.

    Article Title: Neuronal Cell Bodies Remotely Regulate Axonal Growth Response to Localized Netrin-1 Treatment via Second Messenger and DCC Dynamics
    Article Snippet: .. We hypothesized that Netrin-1 evokes calcium efflux through RyRs in the growth cones, which propagates toward the cell bodies and mediates the DCC insertion into their plasma membranes. .. To test this hypothesis, we exposed distal axons to 1.0 μg/ml Netrin-1 for 25 min and simultaneously inhibited their RyRs with 100 μM ryanodine (high Ry).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    R&D Systems netrin 1 blocking
    Cellular distribution of <t>netrin-1</t> in HUVECs. Corrected total (A) and nuclear (B) cell fluorescence of netrin-1 [corrected total cell fluorescence (CTCF) and corrected nuclear cell fluorescence (CNCF), respectively] in HUVECs following different treatments as indicated. The ratio of CTCF to CNCF is also displayed (C). Panel (D) shows representative micrographs (20× magnification fields) of netrin-1 staining (in red) in non-permeabilized (left) and permeabilized (right) HUVECs under the different experimental conditions as specified (scale bars = 10 μm). Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. Concentration of the full-length/secreted isoform of netrin-1 was measured in cell supernatants and is displayed in (E). (F) and (G) report the levels of PGE 2 and TxB 2 , respectively, in HUVEC supernatant following different treatments. n = 3–7. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; SA: salicylic acid, 0.5 mM; TSA, 400 nM; TNF-α, 10 ng·mL –1 . * P
    Netrin 1 Blocking, supplied by R&D Systems, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/netrin 1 blocking/product/R&D Systems
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    netrin 1 blocking - by Bioz Stars, 2020-11
    88/100 stars
      Buy from Supplier

    94
    R&D Systems recombinant mouse netrin 1
    Increased OC formation in Myo10 m/m BMMs in response to <t>netrin-1.</t> (A-D) TRAP staining analysis of OC derived from Myo10 +/+ and Myo10 m/m mice in the presence of control or netrin-1 medium. Representative images are shown in A and B. Bars, 50 μm. Quantitative analysis (mean ± SD, n=8) of the TRAP + multinuclei cell (MNCs) density are presented in C and D. ** p
    Recombinant Mouse Netrin 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant mouse netrin 1/product/R&D Systems
    Average 94 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    recombinant mouse netrin 1 - by Bioz Stars, 2020-11
    94/100 stars
      Buy from Supplier

    91
    R&D Systems rat anti netrin 1
    Cellular distribution of <t>netrin-1</t> in HUVECs. Corrected total (A) and nuclear (B) cell fluorescence of netrin-1 [corrected total cell fluorescence (CTCF) and corrected nuclear cell fluorescence (CNCF), respectively] in HUVECs following different treatments as indicated. The ratio of CTCF to CNCF is also displayed (C). Panel (D) shows representative micrographs (20× magnification fields) of netrin-1 staining (in red) in non-permeabilized (left) and permeabilized (right) HUVECs under the different experimental conditions as specified (scale bars = 10 μm). Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. Concentration of the full-length/secreted isoform of netrin-1 was measured in cell supernatants and is displayed in (E). (F) and (G) report the levels of PGE 2 and TxB 2 , respectively, in HUVEC supernatant following different treatments. n = 3–7. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; SA: salicylic acid, 0.5 mM; TSA, 400 nM; TNF-α, 10 ng·mL –1 . * P
    Rat Anti Netrin 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat anti netrin 1/product/R&D Systems
    Average 91 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    rat anti netrin 1 - by Bioz Stars, 2020-11
    91/100 stars
      Buy from Supplier

    Image Search Results


    Cellular distribution of netrin-1 in HUVECs. Corrected total (A) and nuclear (B) cell fluorescence of netrin-1 [corrected total cell fluorescence (CTCF) and corrected nuclear cell fluorescence (CNCF), respectively] in HUVECs following different treatments as indicated. The ratio of CTCF to CNCF is also displayed (C). Panel (D) shows representative micrographs (20× magnification fields) of netrin-1 staining (in red) in non-permeabilized (left) and permeabilized (right) HUVECs under the different experimental conditions as specified (scale bars = 10 μm). Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. Concentration of the full-length/secreted isoform of netrin-1 was measured in cell supernatants and is displayed in (E). (F) and (G) report the levels of PGE 2 and TxB 2 , respectively, in HUVEC supernatant following different treatments. n = 3–7. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; SA: salicylic acid, 0.5 mM; TSA, 400 nM; TNF-α, 10 ng·mL –1 . * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: Cellular distribution of netrin-1 in HUVECs. Corrected total (A) and nuclear (B) cell fluorescence of netrin-1 [corrected total cell fluorescence (CTCF) and corrected nuclear cell fluorescence (CNCF), respectively] in HUVECs following different treatments as indicated. The ratio of CTCF to CNCF is also displayed (C). Panel (D) shows representative micrographs (20× magnification fields) of netrin-1 staining (in red) in non-permeabilized (left) and permeabilized (right) HUVECs under the different experimental conditions as specified (scale bars = 10 μm). Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. Concentration of the full-length/secreted isoform of netrin-1 was measured in cell supernatants and is displayed in (E). (F) and (G) report the levels of PGE 2 and TxB 2 , respectively, in HUVEC supernatant following different treatments. n = 3–7. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; SA: salicylic acid, 0.5 mM; TSA, 400 nM; TNF-α, 10 ng·mL –1 . * P

    Article Snippet: Experiments were performed in the presence of netrin-1 blocking obtained by i.v. administration of Unc5b-Fc, a netrin-1 receptor, but is fused to the Fc portion of IgG and because of this Unc5b-Fc is referred to as chimera antibody (800 μg·kg−1 body weight; R & D Systems) (Tadagavadi et al ., ).

    Techniques: Fluorescence, Staining, Concentration Assay

    Aspirin (ASA) increases systemic and vascular expression of netrin-1 in ApoE −/− mice. (A) elisa of netrin-1 in plasma, from ApoE − / − mice on 8 week HFD, either untreated (8 weeks; n = 8) or treated with ASA (5 mg·kg − 1 ·day − 1 ; n = 8) or clopidogrel (Clop; 25 mg·kg − 1 ·day − 1 ; n = 8). Also shown in (A) are plasma netrin-1 levels in ApoE − / − mice after 4 weeks of HFD (4 weeks; n = 4) for comparison. (B–C) The micrographs show immunofluorescence staining for netrin-1 (red), the endothelial marker CD31 (green) and nuclei by DAPI (blue) of aortas from ApoE − / − mice after 8 week HFD either untreated (left) or ASA treated (right, ASA 5 mg·kg − 1 ·day − 1 ); magnification = 20× (scale bars = 50 μm). Bottom panels show merging of the three stainings: the white arrows indicate the luminal localization of netrin-1 (red) within the arterial wall and its co-localization with the endothelial cell marker CD31 (green), which is evident in the ASA-treated group but not in either clopidogrel-treated or untreated mice. The graph reports quantification of mean fluorescence intensity for netrin-1 and CD31 in the different groups as specified, and it is reported as fold change versus control (untreated) animals. (D) and (E) report HDAC and HAT activities measured in nuclear extracts isolated from the aortic arterial wall of ApoE − / − mice at the end of the 8 week HFD period, either untreated (8 weeks) or treated with ASA (5 mg·kg − 1 ·day − 1 ) or clopidogrel (25 mg·kg − 1 ·day − 1 ). Also shown in (F) is the HDAC/HAT ratio in the different groups. Data for (A) are shown as median ± interquartile ranges. * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: Aspirin (ASA) increases systemic and vascular expression of netrin-1 in ApoE −/− mice. (A) elisa of netrin-1 in plasma, from ApoE − / − mice on 8 week HFD, either untreated (8 weeks; n = 8) or treated with ASA (5 mg·kg − 1 ·day − 1 ; n = 8) or clopidogrel (Clop; 25 mg·kg − 1 ·day − 1 ; n = 8). Also shown in (A) are plasma netrin-1 levels in ApoE − / − mice after 4 weeks of HFD (4 weeks; n = 4) for comparison. (B–C) The micrographs show immunofluorescence staining for netrin-1 (red), the endothelial marker CD31 (green) and nuclei by DAPI (blue) of aortas from ApoE − / − mice after 8 week HFD either untreated (left) or ASA treated (right, ASA 5 mg·kg − 1 ·day − 1 ); magnification = 20× (scale bars = 50 μm). Bottom panels show merging of the three stainings: the white arrows indicate the luminal localization of netrin-1 (red) within the arterial wall and its co-localization with the endothelial cell marker CD31 (green), which is evident in the ASA-treated group but not in either clopidogrel-treated or untreated mice. The graph reports quantification of mean fluorescence intensity for netrin-1 and CD31 in the different groups as specified, and it is reported as fold change versus control (untreated) animals. (D) and (E) report HDAC and HAT activities measured in nuclear extracts isolated from the aortic arterial wall of ApoE − / − mice at the end of the 8 week HFD period, either untreated (8 weeks) or treated with ASA (5 mg·kg − 1 ·day − 1 ) or clopidogrel (25 mg·kg − 1 ·day − 1 ). Also shown in (F) is the HDAC/HAT ratio in the different groups. Data for (A) are shown as median ± interquartile ranges. * P

    Article Snippet: Experiments were performed in the presence of netrin-1 blocking obtained by i.v. administration of Unc5b-Fc, a netrin-1 receptor, but is fused to the Fc portion of IgG and because of this Unc5b-Fc is referred to as chimera antibody (800 μg·kg−1 body weight; R & D Systems) (Tadagavadi et al ., ).

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Marker, Fluorescence, HAT Assay, Isolation

    Histone 3 acetylation and netrin-1 expression in HUVECs. Western blotting (A, B) and immunofluorescence (C) studies showing degree of histone acetylation in HUVECs in response to different treatments as specified. In the Western blots, densitometric analysis of acetylated H3 (acH3) was normalized to total H3. In immunofluorescence experiments, the corrected nuclear cell fluorescence (CNCF) was calculated (C). Panels (D)–(H) show the effect of aspirin (ASA) on HAT and HDAC activities in HUVECs. HAT (D) and HDAC (E) activities, and HDAC/HAT ratio (F), were measured in nuclear extracts isolated from HUVECs following treatments as specified. Nuclear extracts were isolated from HUVECs not stimulated with TNF-α, and HAT (G) and HDAC (H) activities were tested in the presence of either ASA or salicylic acid. n = 3. ASA, 0.5 mM; SC-560, 30 nM; SA: salicylic acid, 0.5 mM; Indom: indomethacin, 100 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085 (BAY), 5 μM. * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: Histone 3 acetylation and netrin-1 expression in HUVECs. Western blotting (A, B) and immunofluorescence (C) studies showing degree of histone acetylation in HUVECs in response to different treatments as specified. In the Western blots, densitometric analysis of acetylated H3 (acH3) was normalized to total H3. In immunofluorescence experiments, the corrected nuclear cell fluorescence (CNCF) was calculated (C). Panels (D)–(H) show the effect of aspirin (ASA) on HAT and HDAC activities in HUVECs. HAT (D) and HDAC (E) activities, and HDAC/HAT ratio (F), were measured in nuclear extracts isolated from HUVECs following treatments as specified. Nuclear extracts were isolated from HUVECs not stimulated with TNF-α, and HAT (G) and HDAC (H) activities were tested in the presence of either ASA or salicylic acid. n = 3. ASA, 0.5 mM; SC-560, 30 nM; SA: salicylic acid, 0.5 mM; Indom: indomethacin, 100 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085 (BAY), 5 μM. * P

    Article Snippet: Experiments were performed in the presence of netrin-1 blocking obtained by i.v. administration of Unc5b-Fc, a netrin-1 receptor, but is fused to the Fc portion of IgG and because of this Unc5b-Fc is referred to as chimera antibody (800 μg·kg−1 body weight; R & D Systems) (Tadagavadi et al ., ).

    Techniques: Expressing, Western Blot, Immunofluorescence, Fluorescence, HAT Assay, Isolation

    NF-κB activation and gene induction of truncated (nuclear) and full-length (secreted) isoforms of netrin-1. (A) Representative micrographs (20× magnification fields) of p65 staining under the different experimental conditions as indicated. p65 fluorescent images (in green) were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Yellow colour within the nuclei derives from the overlay of p65 signal (green) with nuclear staining (blue), and is indicative of nuclear p65 localization. p65 nuclear translocation was taken as an index of NF-κB activation and is reported in (B) as percentage of nuclei expressing p65 under the different experimental conditions. Panels (C) and (D) show accumulated data for quantitative RT-PCR for netrin-1 (NTN-1) using primers for (C) total netrin-1 (comprising both the full-length and truncated isoforms) or (D) the full-length/secreted isoform specifically, both normalized to the housekeeping gene GAPDH. The ratio of the two isoforms was obtained by normalizing the full-length isoform of netrin-1 to total netrin-1 (E). Data are reported as fold changes compared with control (untreated cells, dotted blue line). n = 3–5. Panels (F) and (G) show representative micrographs (20× magnification fields) of netrin-1 staining (in red) in permeabilized (F) and non-permeabilized (G) HUVECs either untreated (control) or TNF-α treated, either in the presence or absence of the NF-κB inhibitor Bay 11-7085 (BAY), as specified. Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells (F) derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. In permeabilized cells (F), corrected total and nuclear cell fluorescence of netrin-1 (CTCF and CNCF, respectively) following different treatments was calculated and reported in the corresponding graphs. The ratio of CTCF to CNCF is also displayed. Secreted netrin-1 was measured by elisa in cell supernatants and results are reported in (G). n = 3. Scale bars = 10 μm. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085, 5 μM. * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: NF-κB activation and gene induction of truncated (nuclear) and full-length (secreted) isoforms of netrin-1. (A) Representative micrographs (20× magnification fields) of p65 staining under the different experimental conditions as indicated. p65 fluorescent images (in green) were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Yellow colour within the nuclei derives from the overlay of p65 signal (green) with nuclear staining (blue), and is indicative of nuclear p65 localization. p65 nuclear translocation was taken as an index of NF-κB activation and is reported in (B) as percentage of nuclei expressing p65 under the different experimental conditions. Panels (C) and (D) show accumulated data for quantitative RT-PCR for netrin-1 (NTN-1) using primers for (C) total netrin-1 (comprising both the full-length and truncated isoforms) or (D) the full-length/secreted isoform specifically, both normalized to the housekeeping gene GAPDH. The ratio of the two isoforms was obtained by normalizing the full-length isoform of netrin-1 to total netrin-1 (E). Data are reported as fold changes compared with control (untreated cells, dotted blue line). n = 3–5. Panels (F) and (G) show representative micrographs (20× magnification fields) of netrin-1 staining (in red) in permeabilized (F) and non-permeabilized (G) HUVECs either untreated (control) or TNF-α treated, either in the presence or absence of the NF-κB inhibitor Bay 11-7085 (BAY), as specified. Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells (F) derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. In permeabilized cells (F), corrected total and nuclear cell fluorescence of netrin-1 (CTCF and CNCF, respectively) following different treatments was calculated and reported in the corresponding graphs. The ratio of CTCF to CNCF is also displayed. Secreted netrin-1 was measured by elisa in cell supernatants and results are reported in (G). n = 3. Scale bars = 10 μm. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085, 5 μM. * P

    Article Snippet: Experiments were performed in the presence of netrin-1 blocking obtained by i.v. administration of Unc5b-Fc, a netrin-1 receptor, but is fused to the Fc portion of IgG and because of this Unc5b-Fc is referred to as chimera antibody (800 μg·kg−1 body weight; R & D Systems) (Tadagavadi et al ., ).

    Techniques: Activation Assay, Staining, Translocation Assay, Expressing, Quantitative RT-PCR, Fluorescence, Enzyme-linked Immunosorbent Assay

    Increased OC formation in Myo10 m/m BMMs in response to netrin-1. (A-D) TRAP staining analysis of OC derived from Myo10 +/+ and Myo10 m/m mice in the presence of control or netrin-1 medium. Representative images are shown in A and B. Bars, 50 μm. Quantitative analysis (mean ± SD, n=8) of the TRAP + multinuclei cell (MNCs) density are presented in C and D. ** p

    Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

    Article Title: Lack of myosin X enhances osteoclastogenesis and increases cell surface Unc5b in osteoclast-lineage cells

    doi: 10.1002/jbmr.3667

    Figure Lengend Snippet: Increased OC formation in Myo10 m/m BMMs in response to netrin-1. (A-D) TRAP staining analysis of OC derived from Myo10 +/+ and Myo10 m/m mice in the presence of control or netrin-1 medium. Representative images are shown in A and B. Bars, 50 μm. Quantitative analysis (mean ± SD, n=8) of the TRAP + multinuclei cell (MNCs) density are presented in C and D. ** p

    Article Snippet: Recombinant mouse netrin-1 (1109-N1) was purchased from R & D Systems.

    Techniques: Staining, Derivative Assay, Mouse Assay

    Distribution of PhosphoERK1/2 in Control and Netrin-Treated Endoderm and E11.5 Lung ERK1/2 activity is assayed by whole mount immunohistochemistry by using antibody specific for phosphorylated forms of the protein. (A–E) endoderm cultured in Matrigel and 30 ng/ml FGF7 either without (A and D) or with 50 µg/ml Netrin-1 (B) or Netrin-4 (C and E). Note the elevated levels of phosphoERK1/2 in the secondary buds (A and D) but more uniform distribution in Netrin-treated samples (B, C, and E). The focal plane is through the lumen of the cysts in (C) and (I) and near the surface for (A, B, D–F, and H). Inset in (E) shows absence of phosphoERK1/2 in the internalized knobs in Netrin-4-treated samples. (F) Total ERK1/2 is distributed uniformly among budding and interbudding zone. (G) Western blot analysis shows that the relative level of phosphoERK1/2 is decreased in Netrin-4-treated samples. (H and I) In control samples cultured with 250 ng/ml FGF10, localized phosphoERK1/2 is restricted to the tips of elongated secondary buds (H), while Netrin-4-treated endoderm shows a uniform, low level of phosphoERK1/2 activity (I). (J–O) PhosphoERK1/2 activity in normal E11.5 lung. Note that highest ERK1/2 activity is localized to the distal tip of endodermal buds, and initiating buds shows higher phosphoERK than elongating buds (compare arrow and arrowhead in [K]). (L–O) When labeled together with TOTO-3 (blue, to visualize nuclei) and Alexa Fluor 488 phalloidin (green), a sharp boundary is seen between epithelial cells in the dilated distal tip of buds, which show high ERK1/2 activity (red), and the stalk region showing low levels. (M), (N), and (O) correspond to the blue, pink, and white squares in (L), respectively. Scale bar = 100 µm for (A)–(I), 20 µm for (J)–(L), and 10 µm for (M)–(O).

    Journal: Current biology : CB

    Article Title: Novel Role for Netrins in Regulating Epithelial Behavior during Lung Branching Morphogenesis

    doi: 10.1016/j.cub.2004.05.020

    Figure Lengend Snippet: Distribution of PhosphoERK1/2 in Control and Netrin-Treated Endoderm and E11.5 Lung ERK1/2 activity is assayed by whole mount immunohistochemistry by using antibody specific for phosphorylated forms of the protein. (A–E) endoderm cultured in Matrigel and 30 ng/ml FGF7 either without (A and D) or with 50 µg/ml Netrin-1 (B) or Netrin-4 (C and E). Note the elevated levels of phosphoERK1/2 in the secondary buds (A and D) but more uniform distribution in Netrin-treated samples (B, C, and E). The focal plane is through the lumen of the cysts in (C) and (I) and near the surface for (A, B, D–F, and H). Inset in (E) shows absence of phosphoERK1/2 in the internalized knobs in Netrin-4-treated samples. (F) Total ERK1/2 is distributed uniformly among budding and interbudding zone. (G) Western blot analysis shows that the relative level of phosphoERK1/2 is decreased in Netrin-4-treated samples. (H and I) In control samples cultured with 250 ng/ml FGF10, localized phosphoERK1/2 is restricted to the tips of elongated secondary buds (H), while Netrin-4-treated endoderm shows a uniform, low level of phosphoERK1/2 activity (I). (J–O) PhosphoERK1/2 activity in normal E11.5 lung. Note that highest ERK1/2 activity is localized to the distal tip of endodermal buds, and initiating buds shows higher phosphoERK than elongating buds (compare arrow and arrowhead in [K]). (L–O) When labeled together with TOTO-3 (blue, to visualize nuclei) and Alexa Fluor 488 phalloidin (green), a sharp boundary is seen between epithelial cells in the dilated distal tip of buds, which show high ERK1/2 activity (red), and the stalk region showing low levels. (M), (N), and (O) correspond to the blue, pink, and white squares in (L), respectively. Scale bar = 100 µm for (A)–(I), 20 µm for (J)–(L), and 10 µm for (M)–(O).

    Article Snippet: In some experiments, 50 µg/ml recombinant chicken or mouse Netrin-1, mouse or human Netrin-4, mouse Netrin G1a (R & D Systems), or Laminin (Sigma) was mixed with the Matrigel.

    Techniques: Activity Assay, Immunohistochemistry, Cell Culture, Western Blot, Labeling

    Cell Proliferation, Unc5b Expression, and Cell Shape Change after Exogenous Netrin Treatment (A–C) Cell proliferation in control (A) and Netrin-1-treated endoderm (B) or Netrin-4-treated (C) endoderm is estimated by double labeling with antibody to phospho-Histone H3 (red), and Alexa Fluor 488 phalloidin (green), which labels filamentous actin. (D–F) Expression of Unc5b in control (D) and Netrin-1-treated (E) or Netrin-4-treated (F) endoderm examined by whole-mount in situ hybridization. Inset in (D) shows elevated expression in secondary buds. (G–L) Cell shape visualized by double labeling with Alexa Fluor 488 phalloidin (green) and propidium iodide (red). (G–J) In control endoderm, epithelial cells in nonbudding areas are arranged in either a cuboidal ([H], corresponding to approximately the green square in [G]) or pseudostratified layer ([I], blue square in [G]). (J) In the secondary bud (pink square in [G]), cells have an elongated wedge shape and basally localized nuclei. (K and L) In Netrin-4-treated samples, cells in the internal knobs have an irregular shape (L). Scale bar = 100 µm for (A–G) and (K), 10 µm for other panels.

    Journal: Current biology : CB

    Article Title: Novel Role for Netrins in Regulating Epithelial Behavior during Lung Branching Morphogenesis

    doi: 10.1016/j.cub.2004.05.020

    Figure Lengend Snippet: Cell Proliferation, Unc5b Expression, and Cell Shape Change after Exogenous Netrin Treatment (A–C) Cell proliferation in control (A) and Netrin-1-treated endoderm (B) or Netrin-4-treated (C) endoderm is estimated by double labeling with antibody to phospho-Histone H3 (red), and Alexa Fluor 488 phalloidin (green), which labels filamentous actin. (D–F) Expression of Unc5b in control (D) and Netrin-1-treated (E) or Netrin-4-treated (F) endoderm examined by whole-mount in situ hybridization. Inset in (D) shows elevated expression in secondary buds. (G–L) Cell shape visualized by double labeling with Alexa Fluor 488 phalloidin (green) and propidium iodide (red). (G–J) In control endoderm, epithelial cells in nonbudding areas are arranged in either a cuboidal ([H], corresponding to approximately the green square in [G]) or pseudostratified layer ([I], blue square in [G]). (J) In the secondary bud (pink square in [G]), cells have an elongated wedge shape and basally localized nuclei. (K and L) In Netrin-4-treated samples, cells in the internal knobs have an irregular shape (L). Scale bar = 100 µm for (A–G) and (K), 10 µm for other panels.

    Article Snippet: In some experiments, 50 µg/ml recombinant chicken or mouse Netrin-1, mouse or human Netrin-4, mouse Netrin G1a (R & D Systems), or Laminin (Sigma) was mixed with the Matrigel.

    Techniques: Expressing, Labeling, In Situ Hybridization

    A Model for the Possible Roles Played by Netrins during Lung Epithelial Branching Morphogenesis (A) Netrin-1 and -4 made by epithelial cells are deposited at the basement membrane and/or bind locally to epithelial cells at the neck region of elongating endoderm buds. Here they act through Unc5b or an unknown receptor to decrease the local ERK1/2 activity in the endoderm, thus facilitating the outgrowth of buds toward the source of FGF10 in the mesoderm, as well as preventing the generation of ectopic buds. According to this model, the basal lamina plus Netrins acts as a kind of sleeve or “corset,” restricting the morphogenesis of the emerging bud. (B) Our in vitro assays in which endoderm is surrounded by basal lamina components in the Matrigel mimic the effect of localized, ectopic Netrins at the distal tip, thus preventing bud outgrowth. In contrast, some cells accumulate at the inside of the lumen and form internal knobs.

    Journal: Current biology : CB

    Article Title: Novel Role for Netrins in Regulating Epithelial Behavior during Lung Branching Morphogenesis

    doi: 10.1016/j.cub.2004.05.020

    Figure Lengend Snippet: A Model for the Possible Roles Played by Netrins during Lung Epithelial Branching Morphogenesis (A) Netrin-1 and -4 made by epithelial cells are deposited at the basement membrane and/or bind locally to epithelial cells at the neck region of elongating endoderm buds. Here they act through Unc5b or an unknown receptor to decrease the local ERK1/2 activity in the endoderm, thus facilitating the outgrowth of buds toward the source of FGF10 in the mesoderm, as well as preventing the generation of ectopic buds. According to this model, the basal lamina plus Netrins acts as a kind of sleeve or “corset,” restricting the morphogenesis of the emerging bud. (B) Our in vitro assays in which endoderm is surrounded by basal lamina components in the Matrigel mimic the effect of localized, ectopic Netrins at the distal tip, thus preventing bud outgrowth. In contrast, some cells accumulate at the inside of the lumen and form internal knobs.

    Article Snippet: In some experiments, 50 µg/ml recombinant chicken or mouse Netrin-1, mouse or human Netrin-4, mouse Netrin G1a (R & D Systems), or Laminin (Sigma) was mixed with the Matrigel.

    Techniques: Activated Clotting Time Assay, Activity Assay, In Vitro

    Spatial and Temporal Expression of Genes Encoding Netrins and Their Receptors during Lung Branching Morphogenesis (A)–(F), (H)–(J), and (M) are the results of whole mount and (G and N) radioactive section in situ hybridization.  netrin 1  is expressed at E10.5 (A), continues at E11.5 (B and C), E12.5 (D and E), and E13.5 (F) but dramatically declines by E15.5 (G). Transcripts are present in the proximal epithelium and enriched in the stalks of buds but are excluded from the dilated distal tips (arrows in [B], [C], and [E]). (C) and (E) are enlargements of (B) and (D), respectively. Discrete regions of distal mesoderm in the right accessory and medial lobes also show  netrin 1  expression (arrowheads in [B], [C], and [E]). (H)  netrin 3  is expressed diffusely at low levels throughout the lung endoderm and mesoderm at E11.5.  netrin 4  is expressed in proximal, but not distal, endoderm (arrows) at E11.5 (I) and E12.5 (J). (K and L) At E11.5, whole-mount immunohistochemistry localized DCC basally in the proximal endoderm (arrows) and apically in distal epithelium at the tips of buds (arrowhead in [L]). (K) and (L) correspond to the approximate positions of the pink and yellow squares in (B). (M and N)  Unc5b  transcripts are present in distal endoderm (arrows) and mesenchyme, but not in proximal endoderm (arrowhead) at E12.5 (M) and E13.5 (N). Inset in (M) shows that  Unc5b  is expressed in distal tip and neck region of endoderm. Scale bar = 100 µm.

    Journal: Current biology : CB

    Article Title: Novel Role for Netrins in Regulating Epithelial Behavior during Lung Branching Morphogenesis

    doi: 10.1016/j.cub.2004.05.020

    Figure Lengend Snippet: Spatial and Temporal Expression of Genes Encoding Netrins and Their Receptors during Lung Branching Morphogenesis (A)–(F), (H)–(J), and (M) are the results of whole mount and (G and N) radioactive section in situ hybridization. netrin 1 is expressed at E10.5 (A), continues at E11.5 (B and C), E12.5 (D and E), and E13.5 (F) but dramatically declines by E15.5 (G). Transcripts are present in the proximal epithelium and enriched in the stalks of buds but are excluded from the dilated distal tips (arrows in [B], [C], and [E]). (C) and (E) are enlargements of (B) and (D), respectively. Discrete regions of distal mesoderm in the right accessory and medial lobes also show netrin 1 expression (arrowheads in [B], [C], and [E]). (H) netrin 3 is expressed diffusely at low levels throughout the lung endoderm and mesoderm at E11.5. netrin 4 is expressed in proximal, but not distal, endoderm (arrows) at E11.5 (I) and E12.5 (J). (K and L) At E11.5, whole-mount immunohistochemistry localized DCC basally in the proximal endoderm (arrows) and apically in distal epithelium at the tips of buds (arrowhead in [L]). (K) and (L) correspond to the approximate positions of the pink and yellow squares in (B). (M and N) Unc5b transcripts are present in distal endoderm (arrows) and mesenchyme, but not in proximal endoderm (arrowhead) at E12.5 (M) and E13.5 (N). Inset in (M) shows that Unc5b is expressed in distal tip and neck region of endoderm. Scale bar = 100 µm.

    Article Snippet: In some experiments, 50 µg/ml recombinant chicken or mouse Netrin-1, mouse or human Netrin-4, mouse Netrin G1a (R & D Systems), or Laminin (Sigma) was mixed with the Matrigel.

    Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Droplet Countercurrent Chromatography

    Cellular distribution of netrin-1 in HUVECs. Corrected total (A) and nuclear (B) cell fluorescence of netrin-1 [corrected total cell fluorescence (CTCF) and corrected nuclear cell fluorescence (CNCF), respectively] in HUVECs following different treatments as indicated. The ratio of CTCF to CNCF is also displayed (C). Panel (D) shows representative micrographs (20× magnification fields) of netrin-1 staining (in red) in non-permeabilized (left) and permeabilized (right) HUVECs under the different experimental conditions as specified (scale bars = 10 μm). Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. Concentration of the full-length/secreted isoform of netrin-1 was measured in cell supernatants and is displayed in (E). (F) and (G) report the levels of PGE 2 and TxB 2 , respectively, in HUVEC supernatant following different treatments. n = 3–7. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; SA: salicylic acid, 0.5 mM; TSA, 400 nM; TNF-α, 10 ng·mL –1 . * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: Cellular distribution of netrin-1 in HUVECs. Corrected total (A) and nuclear (B) cell fluorescence of netrin-1 [corrected total cell fluorescence (CTCF) and corrected nuclear cell fluorescence (CNCF), respectively] in HUVECs following different treatments as indicated. The ratio of CTCF to CNCF is also displayed (C). Panel (D) shows representative micrographs (20× magnification fields) of netrin-1 staining (in red) in non-permeabilized (left) and permeabilized (right) HUVECs under the different experimental conditions as specified (scale bars = 10 μm). Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. Concentration of the full-length/secreted isoform of netrin-1 was measured in cell supernatants and is displayed in (E). (F) and (G) report the levels of PGE 2 and TxB 2 , respectively, in HUVEC supernatant following different treatments. n = 3–7. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; SA: salicylic acid, 0.5 mM; TSA, 400 nM; TNF-α, 10 ng·mL –1 . * P

    Article Snippet: Immunofluorescence staining was performed using rat anti-netrin-1 (1:200; R & D System, Abingdon, UK), rabbit anti-acetylated histone (1:400; Cell Signalling, Hitchin, UK) and rabbit anti-p65 (1:100; Abcam, Cambridge, UK).

    Techniques: Fluorescence, Staining, Concentration Assay

    Aspirin (ASA) increases systemic and vascular expression of netrin-1 in ApoE −/− mice. (A) elisa of netrin-1 in plasma, from ApoE − / − mice on 8 week HFD, either untreated (8 weeks; n = 8) or treated with ASA (5 mg·kg − 1 ·day − 1 ; n = 8) or clopidogrel (Clop; 25 mg·kg − 1 ·day − 1 ; n = 8). Also shown in (A) are plasma netrin-1 levels in ApoE − / − mice after 4 weeks of HFD (4 weeks; n = 4) for comparison. (B–C) The micrographs show immunofluorescence staining for netrin-1 (red), the endothelial marker CD31 (green) and nuclei by DAPI (blue) of aortas from ApoE − / − mice after 8 week HFD either untreated (left) or ASA treated (right, ASA 5 mg·kg − 1 ·day − 1 ); magnification = 20× (scale bars = 50 μm). Bottom panels show merging of the three stainings: the white arrows indicate the luminal localization of netrin-1 (red) within the arterial wall and its co-localization with the endothelial cell marker CD31 (green), which is evident in the ASA-treated group but not in either clopidogrel-treated or untreated mice. The graph reports quantification of mean fluorescence intensity for netrin-1 and CD31 in the different groups as specified, and it is reported as fold change versus control (untreated) animals. (D) and (E) report HDAC and HAT activities measured in nuclear extracts isolated from the aortic arterial wall of ApoE − / − mice at the end of the 8 week HFD period, either untreated (8 weeks) or treated with ASA (5 mg·kg − 1 ·day − 1 ) or clopidogrel (25 mg·kg − 1 ·day − 1 ). Also shown in (F) is the HDAC/HAT ratio in the different groups. Data for (A) are shown as median ± interquartile ranges. * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: Aspirin (ASA) increases systemic and vascular expression of netrin-1 in ApoE −/− mice. (A) elisa of netrin-1 in plasma, from ApoE − / − mice on 8 week HFD, either untreated (8 weeks; n = 8) or treated with ASA (5 mg·kg − 1 ·day − 1 ; n = 8) or clopidogrel (Clop; 25 mg·kg − 1 ·day − 1 ; n = 8). Also shown in (A) are plasma netrin-1 levels in ApoE − / − mice after 4 weeks of HFD (4 weeks; n = 4) for comparison. (B–C) The micrographs show immunofluorescence staining for netrin-1 (red), the endothelial marker CD31 (green) and nuclei by DAPI (blue) of aortas from ApoE − / − mice after 8 week HFD either untreated (left) or ASA treated (right, ASA 5 mg·kg − 1 ·day − 1 ); magnification = 20× (scale bars = 50 μm). Bottom panels show merging of the three stainings: the white arrows indicate the luminal localization of netrin-1 (red) within the arterial wall and its co-localization with the endothelial cell marker CD31 (green), which is evident in the ASA-treated group but not in either clopidogrel-treated or untreated mice. The graph reports quantification of mean fluorescence intensity for netrin-1 and CD31 in the different groups as specified, and it is reported as fold change versus control (untreated) animals. (D) and (E) report HDAC and HAT activities measured in nuclear extracts isolated from the aortic arterial wall of ApoE − / − mice at the end of the 8 week HFD period, either untreated (8 weeks) or treated with ASA (5 mg·kg − 1 ·day − 1 ) or clopidogrel (25 mg·kg − 1 ·day − 1 ). Also shown in (F) is the HDAC/HAT ratio in the different groups. Data for (A) are shown as median ± interquartile ranges. * P

    Article Snippet: Immunofluorescence staining was performed using rat anti-netrin-1 (1:200; R & D System, Abingdon, UK), rabbit anti-acetylated histone (1:400; Cell Signalling, Hitchin, UK) and rabbit anti-p65 (1:100; Abcam, Cambridge, UK).

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Marker, Fluorescence, HAT Assay, Isolation

    Histone 3 acetylation and netrin-1 expression in HUVECs. Western blotting (A, B) and immunofluorescence (C) studies showing degree of histone acetylation in HUVECs in response to different treatments as specified. In the Western blots, densitometric analysis of acetylated H3 (acH3) was normalized to total H3. In immunofluorescence experiments, the corrected nuclear cell fluorescence (CNCF) was calculated (C). Panels (D)–(H) show the effect of aspirin (ASA) on HAT and HDAC activities in HUVECs. HAT (D) and HDAC (E) activities, and HDAC/HAT ratio (F), were measured in nuclear extracts isolated from HUVECs following treatments as specified. Nuclear extracts were isolated from HUVECs not stimulated with TNF-α, and HAT (G) and HDAC (H) activities were tested in the presence of either ASA or salicylic acid. n = 3. ASA, 0.5 mM; SC-560, 30 nM; SA: salicylic acid, 0.5 mM; Indom: indomethacin, 100 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085 (BAY), 5 μM. * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: Histone 3 acetylation and netrin-1 expression in HUVECs. Western blotting (A, B) and immunofluorescence (C) studies showing degree of histone acetylation in HUVECs in response to different treatments as specified. In the Western blots, densitometric analysis of acetylated H3 (acH3) was normalized to total H3. In immunofluorescence experiments, the corrected nuclear cell fluorescence (CNCF) was calculated (C). Panels (D)–(H) show the effect of aspirin (ASA) on HAT and HDAC activities in HUVECs. HAT (D) and HDAC (E) activities, and HDAC/HAT ratio (F), were measured in nuclear extracts isolated from HUVECs following treatments as specified. Nuclear extracts were isolated from HUVECs not stimulated with TNF-α, and HAT (G) and HDAC (H) activities were tested in the presence of either ASA or salicylic acid. n = 3. ASA, 0.5 mM; SC-560, 30 nM; SA: salicylic acid, 0.5 mM; Indom: indomethacin, 100 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085 (BAY), 5 μM. * P

    Article Snippet: Immunofluorescence staining was performed using rat anti-netrin-1 (1:200; R & D System, Abingdon, UK), rabbit anti-acetylated histone (1:400; Cell Signalling, Hitchin, UK) and rabbit anti-p65 (1:100; Abcam, Cambridge, UK).

    Techniques: Expressing, Western Blot, Immunofluorescence, Fluorescence, HAT Assay, Isolation

    NF-κB activation and gene induction of truncated (nuclear) and full-length (secreted) isoforms of netrin-1. (A) Representative micrographs (20× magnification fields) of p65 staining under the different experimental conditions as indicated. p65 fluorescent images (in green) were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Yellow colour within the nuclei derives from the overlay of p65 signal (green) with nuclear staining (blue), and is indicative of nuclear p65 localization. p65 nuclear translocation was taken as an index of NF-κB activation and is reported in (B) as percentage of nuclei expressing p65 under the different experimental conditions. Panels (C) and (D) show accumulated data for quantitative RT-PCR for netrin-1 (NTN-1) using primers for (C) total netrin-1 (comprising both the full-length and truncated isoforms) or (D) the full-length/secreted isoform specifically, both normalized to the housekeeping gene GAPDH. The ratio of the two isoforms was obtained by normalizing the full-length isoform of netrin-1 to total netrin-1 (E). Data are reported as fold changes compared with control (untreated cells, dotted blue line). n = 3–5. Panels (F) and (G) show representative micrographs (20× magnification fields) of netrin-1 staining (in red) in permeabilized (F) and non-permeabilized (G) HUVECs either untreated (control) or TNF-α treated, either in the presence or absence of the NF-κB inhibitor Bay 11-7085 (BAY), as specified. Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells (F) derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. In permeabilized cells (F), corrected total and nuclear cell fluorescence of netrin-1 (CTCF and CNCF, respectively) following different treatments was calculated and reported in the corresponding graphs. The ratio of CTCF to CNCF is also displayed. Secreted netrin-1 was measured by elisa in cell supernatants and results are reported in (G). n = 3. Scale bars = 10 μm. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085, 5 μM. * P

    Journal: British Journal of Pharmacology

    Article Title: Aspirin-induced histone acetylation in endothelial cells enhances synthesis of the secreted isoform of netrin-1 thus inhibiting monocyte vascular infiltration

    doi: 10.1111/bph.13144

    Figure Lengend Snippet: NF-κB activation and gene induction of truncated (nuclear) and full-length (secreted) isoforms of netrin-1. (A) Representative micrographs (20× magnification fields) of p65 staining under the different experimental conditions as indicated. p65 fluorescent images (in green) were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Yellow colour within the nuclei derives from the overlay of p65 signal (green) with nuclear staining (blue), and is indicative of nuclear p65 localization. p65 nuclear translocation was taken as an index of NF-κB activation and is reported in (B) as percentage of nuclei expressing p65 under the different experimental conditions. Panels (C) and (D) show accumulated data for quantitative RT-PCR for netrin-1 (NTN-1) using primers for (C) total netrin-1 (comprising both the full-length and truncated isoforms) or (D) the full-length/secreted isoform specifically, both normalized to the housekeeping gene GAPDH. The ratio of the two isoforms was obtained by normalizing the full-length isoform of netrin-1 to total netrin-1 (E). Data are reported as fold changes compared with control (untreated cells, dotted blue line). n = 3–5. Panels (F) and (G) show representative micrographs (20× magnification fields) of netrin-1 staining (in red) in permeabilized (F) and non-permeabilized (G) HUVECs either untreated (control) or TNF-α treated, either in the presence or absence of the NF-κB inhibitor Bay 11-7085 (BAY), as specified. Netrin-1 fluorescent images were merged with corresponding fluorescent nuclear staining with Hoechst (in blue). Pink colour within the nuclei in permeabilized cells (F) derives from the overlay of netrin-1 signal (red) with nuclear staining (blue), and is indicative of nuclear netrin-1 localization. In permeabilized cells (F), corrected total and nuclear cell fluorescence of netrin-1 (CTCF and CNCF, respectively) following different treatments was calculated and reported in the corresponding graphs. The ratio of CTCF to CNCF is also displayed. Secreted netrin-1 was measured by elisa in cell supernatants and results are reported in (G). n = 3. Scale bars = 10 μm. ASA: aspirin, 0.5 mM; SC-560, 30 nM; Indom: indomethacin, 100 μM; NS-398, 10 μM; TSA, 400 nM; TNF-α, 10 ng·mL −1 ; Bay 11-7085, 5 μM. * P

    Article Snippet: Immunofluorescence staining was performed using rat anti-netrin-1 (1:200; R & D System, Abingdon, UK), rabbit anti-acetylated histone (1:400; Cell Signalling, Hitchin, UK) and rabbit anti-p65 (1:100; Abcam, Cambridge, UK).

    Techniques: Activation Assay, Staining, Translocation Assay, Expressing, Quantitative RT-PCR, Fluorescence, Enzyme-linked Immunosorbent Assay