ngf neutralizing antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs ngf neutralizing antibody
    <t>ENT-A013,</t> ENT-A013E and ENT-A013Z protect PC12 cells against serum deprivation-induced cell death in a TrkA-dependent manner. PC12 cells were starved of serum and treated with <t>NGF,</t> ENT-A013, ENT-A013E, ENT-A013Z or vehicle in the presence or absence of a specific TrkA inhibitor GW441756 for 24 h and CellTox assay was used to quantify cell death. CellTox positive cell number was normalized to total Hoescht positive cell number in each image. Representative images from 4–9 independent experiments are shown ( A ) and quantification ( B ). Data represent ±SEM. One-way ANOVA, Tukey’s test correction: ns: non-significant; * and # p
    Ngf Neutralizing Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ngf neutralizing antibody/product/Alomone Labs
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ngf neutralizing antibody - by Bioz Stars, 2022-08
    94/100 stars

    Images

    1) Product Images from "Development and Biological Characterization of a Novel Selective TrkA Agonist with Neuroprotective Properties against Amyloid Toxicity"

    Article Title: Development and Biological Characterization of a Novel Selective TrkA Agonist with Neuroprotective Properties against Amyloid Toxicity

    Journal: Biomedicines

    doi: 10.3390/biomedicines10030614

    ENT-A013, ENT-A013E and ENT-A013Z protect PC12 cells against serum deprivation-induced cell death in a TrkA-dependent manner. PC12 cells were starved of serum and treated with NGF, ENT-A013, ENT-A013E, ENT-A013Z or vehicle in the presence or absence of a specific TrkA inhibitor GW441756 for 24 h and CellTox assay was used to quantify cell death. CellTox positive cell number was normalized to total Hoescht positive cell number in each image. Representative images from 4–9 independent experiments are shown ( A ) and quantification ( B ). Data represent ±SEM. One-way ANOVA, Tukey’s test correction: ns: non-significant; * and # p
    Figure Legend Snippet: ENT-A013, ENT-A013E and ENT-A013Z protect PC12 cells against serum deprivation-induced cell death in a TrkA-dependent manner. PC12 cells were starved of serum and treated with NGF, ENT-A013, ENT-A013E, ENT-A013Z or vehicle in the presence or absence of a specific TrkA inhibitor GW441756 for 24 h and CellTox assay was used to quantify cell death. CellTox positive cell number was normalized to total Hoescht positive cell number in each image. Representative images from 4–9 independent experiments are shown ( A ) and quantification ( B ). Data represent ±SEM. One-way ANOVA, Tukey’s test correction: ns: non-significant; * and # p

    Techniques Used:

    Docking poses of the two ENT-A013 isomers, ENT-A013E and ENT-A013Z, in sites 1a and 1b at the two symmetry-related TrkA-D5—NGF interfaces of the neurotrophin-receptor complex. The close-up views show residues lining the two sites. TrkA-D5 and NGF are shown in green and blue cartoon representation, respectively, with selected residues in stick representation colored by atom type. The compounds are shown in orange stick representation.
    Figure Legend Snippet: Docking poses of the two ENT-A013 isomers, ENT-A013E and ENT-A013Z, in sites 1a and 1b at the two symmetry-related TrkA-D5—NGF interfaces of the neurotrophin-receptor complex. The close-up views show residues lining the two sites. TrkA-D5 and NGF are shown in green and blue cartoon representation, respectively, with selected residues in stick representation colored by atom type. The compounds are shown in orange stick representation.

    Techniques Used:

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    Alomone Labs mouse anti ngf neutralizing antibodies
    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of <t>NGF</t> before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag <t>MFG-E8</t> D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P
    Mouse Anti Ngf Neutralizing Antibodies, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti ngf neutralizing antibodies/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti ngf neutralizing antibodies - by Bioz Stars, 2022-08
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    92
    Alomone Labs ngf prongf neutralizing antibody
    Effect of <t>proNGF</t> administration and <t>anti-NGF/proNGF</t> addition on SCDM differentiation. ( A ) SCDMs were allowed to differentiate into myotubes in the differentiation medium (DM), in presence or not of proNGF (100 ng/mL) for 72 h. Cells were observed with bright field microscopy and three images for every single experiment were captured. ( B ) SCDMs were treated with anti-NGF/proNGF (500 ng/mL) and induced to differentiate in DM for 72 h. Three images for each experimental group were captured under a brightfield microscope, and then analyzed to evaluate myotube length, myotube diameter and myotube number. ( C ) SCDMs were induced to differentiate in the presence of proNGF (100 ng/mL) or anti-NGF/proNGF (500 ng/mL) for 72 h. Once differentiation was completed, the cells were fixed in paraformaldehyde (PFA) 4% and incubated overnight with anti-MyHC (green) to evaluate the fusion index by immunofluorescence. DAPI (blue) was used to counterstain the nuclei. Fusion index was calculated as the percentage of nuclei incorporated into myotubes on the total nuclei present in each field. All the experiments consisted of at least three biological replicates. Scale bars: 100 μm. Values are expressed as the mean ± SD. Statistical analysis was performed by using the Student’s t test. * p
    Ngf Prongf Neutralizing Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ngf prongf neutralizing antibody/product/Alomone Labs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ngf prongf neutralizing antibody - by Bioz Stars, 2022-08
    92/100 stars
      Buy from Supplier

    Image Search Results


    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Activity Assay, Cell Culture, Staining

    NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Staining

    PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, In Vitro, Staining

    Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Purification, Labeling

    Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Blocking Assay, Cell Culture, Staining

    Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Staining

    Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Inhibiting caspase activity prevents PS exposure only in apoptotic-dependent axon degeneration. a DRG axons were cultured for 48 h in the presence of NGF before a 24 h treatment with the pan-caspase inhibitor Z-VAD (50 μM) or vehicle (DMSO) with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. Z-VAD had no effect on basal levels of PS exposure. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with DMSO or 50 μM Z-VAD. PS exposure was significantly reduced after 16 h and 24 h of Z-VAD treatment. d , e DRG axons were cultured for 96 h and then treated with 40 nM vincristine with DMSO or Z-VAD for 8, 16, or 24 h. Z-VAD treatment did not prevent PS exposure on vincristine-treated axons at any time point. f , g DRG axons were cultured for 48 h before axons were axotomized using a sharp needle and cultured with DMSO or Z-VAD for 4, 8, or 16 h. Z-VAD treatment did not prevent PS exposure on axotomized axons at any time point. Error bars indicate mean ± SEM, p -value (two-way ANOVA): *** P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Activity Assay, Cell Culture, Staining

    NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: NAD + supplementation suppresses PS exposure on degenerating axons. a DRG axons were cultured for 48 h in the presence of NGF before supplementation with 20 mM NAD + or vehicle control and an additional 24 h of culture, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. NAD + had no effect on basal PS exposure levels. b , c DRG axons were cultured for 48 h and then NGF-deprived for 8, 16, or 24 h with vehicle control or 20 mM NAD + supplement. PS exposure was reduced significantly after 16 h in the NAD + treated axons, but not at 24 h. d , e DRG axons were cultured for 96 h before treatment with 40 nM vincristine with vehicle or 120 mM NAD + supplement for 8, 16, or 24 h. PS exposure was reduced significantly after 16 h and 24 h. f , g DRG axons were cultured for 48 h before axons were axotomized using sharp needle, and cultured with vehicle or 20 mM NAD + supplement for 4, 8, or 16 h. NAD + supplement prevented PS exposure on axotomized axons in all tested times. h ATP levels with or without NAD + supplement. DRG explants were cultured on cell inserts for 48 h before treated with NGF deprivation, vincristine or axotomy. After indicated time points, axonal compartment were collected and ATP levels were quantified. All three treatments significantly reduced axonal ATP levels, compare with control. NAD + supplement prevented ATP reduction and rescued axonal ATP levels back to control levels. i DRG axons were cultured for 48 h and then treated with 10 μM FK866 or DMSO for 5, 10 and 24 h. PS exposure was not affected by FK866 treatment at all time point tested. j Quntification of PS exspoure levels after FK866 or DMSO treatment. Error bars indicate mean ± SEM, p -value (two-way ANOVA): * P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Staining

    PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: PS is exposed on sub-axonal segments. a Schematic representation of microfluidic chambers: axons and cell bodies are in separate compartments, allowing selective treatment of the axonal compartment. Dissociated tdTomato-positive DRG neurons were cultured in microfluidic chambers. After 5 days in vitro (DIV), the axonal compartment was treated, as indicated, for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. b In control untreated cultures, the axons and cell bodies remained intact, and PS was not detected on the outer membrane. c , d Local axonal degeneration induced by NGF deprivation ( c ) or 40 nM vincristine treatment ( d ) for 24 h resulted in PS exposure on the treated distal axonal segment but not on the soma/proximal axons. e Quantification of PS exposure levels on the soma and axonal compartment in control and local axon degeneration. Error bars show mean ± SEM, p -value (student t test): * P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, In Vitro, Staining

    Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Masking PS signal reduces axonal debris engulfment. a , b tdTomato-positive DRG neurons were cultured in MFC for 5 days or as explants for 48 h, before being NGF-deprived for 24 h or axotomized for 16 h (Figure show NGF-deprived axons), with ( b ) or without ( a ) 10 μg/ml purified flag MFG-E8 D89E . White arrowheads marks Necl4/Td tomato double positive cells. c Quantification of percentage of engulfing glia cells. To evaluate engulfment of axonal debris, we counted labeled cells to determine the percentage of Necl4-positive/TdTomato debris-positive glia cells as a fraction of all Necl4-positive cells. Error bars mean ± SEM, p -value (student t test): ***p

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Purification, Labeling

    Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Blocking extracellular Ca ++ influx does not prevent PS exposure. a DRG axons were cultured for 48 h in the presence of NGF before treatment with 2 mM EGTA for 24 h, with addition of flag MFG-E8 D89E . After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. EGTA had no effect on the basal levels of PS exposure. b-g DRG axons were cultured for 48 h and then treated as indicated in the presence of vehicle (upper rows) or 2 mM EGTA (lower rows). b , c EGTA had no effect on the exposure of PS in DRG axons deprived of NGF for 8, 16, or 24 h. d , e EGTA had no effect on the exposure of PS in DRG axons treated with vincristine for 8, 16, or 24 h. f , g EGTA had no effect on the exposure of PS in axotomized DRG axons at 4, 8, or 16 h post-axotomy; however, it completely protected axons from degeneration. c , e , g Error bars indicate mean ± SEM, significance determined by two-way ANOVA, Scale bar: 100 μm, N = minimum of five separate explants were analyzed per experimental condition

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Blocking Assay, Cell Culture, Staining

    Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P

    Journal: Cell Death & Disease

    Article Title: Phosphatidylserine is a marker for axonal debris engulfment but its exposure can be decoupled from degeneration

    doi: 10.1038/s41419-018-1155-z

    Figure Lengend Snippet: Early activators of axonal degeneration control PS exposure. a Schematic representation of the pathways that control axonal degeneration. Key activators of each pathway, as well as other downstream contributors to the pathways are depicted. Pharmacological treatments used in the experiments are marked in purple. b-d DRG explants of WT ( b ), Bax -/- ( c ) and Sarm1 -/- ( d ) embryos were cultured for 48 to 96 h in the presence of NGF before axon degeneration was initiated by NGF deprivation, 40 nM vincristine, or axotomy, with addition of flag MFG-E8 D89E , for additional 16 h (Axotomy) or 24 h (NGF deprivation and Vincristine). After treatment, cells were briefly fixed, stained with anti-Flag, and PS exposure was measured by anti-Flag staining intensity. WT axons expose PS after all treatments, while Bax null axons expose PS after vincristine and axotomy, but not after NGF deprivation. Sarm1 null axons expose PS after NGF deprivation but not after vincristine or axotomy. e Quantification of PS exposure levels on WT, Bax -/- and Sarm1 -/- axons in all treatments. Error bars mean ± SEM, p -value, compare with WT exposure levels (student t test): *** P

    Article Snippet: For NGF deprivation, the medium was exchanged for medium lacking NGF with addition of 1 mg/ml of mouse anti-NGF neutralizing antibodies (Alomone Labs; AN-240) and Flag MFG-EE8D89E .

    Techniques: Cell Culture, Staining

    ENT-A013, ENT-A013E and ENT-A013Z protect PC12 cells against serum deprivation-induced cell death in a TrkA-dependent manner. PC12 cells were starved of serum and treated with NGF, ENT-A013, ENT-A013E, ENT-A013Z or vehicle in the presence or absence of a specific TrkA inhibitor GW441756 for 24 h and CellTox assay was used to quantify cell death. CellTox positive cell number was normalized to total Hoescht positive cell number in each image. Representative images from 4–9 independent experiments are shown ( A ) and quantification ( B ). Data represent ±SEM. One-way ANOVA, Tukey’s test correction: ns: non-significant; * and # p

    Journal: Biomedicines

    Article Title: Development and Biological Characterization of a Novel Selective TrkA Agonist with Neuroprotective Properties against Amyloid Toxicity

    doi: 10.3390/biomedicines10030614

    Figure Lengend Snippet: ENT-A013, ENT-A013E and ENT-A013Z protect PC12 cells against serum deprivation-induced cell death in a TrkA-dependent manner. PC12 cells were starved of serum and treated with NGF, ENT-A013, ENT-A013E, ENT-A013Z or vehicle in the presence or absence of a specific TrkA inhibitor GW441756 for 24 h and CellTox assay was used to quantify cell death. CellTox positive cell number was normalized to total Hoescht positive cell number in each image. Representative images from 4–9 independent experiments are shown ( A ) and quantification ( B ). Data represent ±SEM. One-way ANOVA, Tukey’s test correction: ns: non-significant; * and # p

    Article Snippet: For neurite outgrowth assay, cells were cultured for 5 days in vitro with medium deprived of NGF in the presence of ENT-A013 (500 nM, every 48 h), along with NGF-neutralizing antibody.

    Techniques:

    Docking poses of the two ENT-A013 isomers, ENT-A013E and ENT-A013Z, in sites 1a and 1b at the two symmetry-related TrkA-D5—NGF interfaces of the neurotrophin-receptor complex. The close-up views show residues lining the two sites. TrkA-D5 and NGF are shown in green and blue cartoon representation, respectively, with selected residues in stick representation colored by atom type. The compounds are shown in orange stick representation.

    Journal: Biomedicines

    Article Title: Development and Biological Characterization of a Novel Selective TrkA Agonist with Neuroprotective Properties against Amyloid Toxicity

    doi: 10.3390/biomedicines10030614

    Figure Lengend Snippet: Docking poses of the two ENT-A013 isomers, ENT-A013E and ENT-A013Z, in sites 1a and 1b at the two symmetry-related TrkA-D5—NGF interfaces of the neurotrophin-receptor complex. The close-up views show residues lining the two sites. TrkA-D5 and NGF are shown in green and blue cartoon representation, respectively, with selected residues in stick representation colored by atom type. The compounds are shown in orange stick representation.

    Article Snippet: For neurite outgrowth assay, cells were cultured for 5 days in vitro with medium deprived of NGF in the presence of ENT-A013 (500 nM, every 48 h), along with NGF-neutralizing antibody.

    Techniques:

    Effect of proNGF administration and anti-NGF/proNGF addition on SCDM differentiation. ( A ) SCDMs were allowed to differentiate into myotubes in the differentiation medium (DM), in presence or not of proNGF (100 ng/mL) for 72 h. Cells were observed with bright field microscopy and three images for every single experiment were captured. ( B ) SCDMs were treated with anti-NGF/proNGF (500 ng/mL) and induced to differentiate in DM for 72 h. Three images for each experimental group were captured under a brightfield microscope, and then analyzed to evaluate myotube length, myotube diameter and myotube number. ( C ) SCDMs were induced to differentiate in the presence of proNGF (100 ng/mL) or anti-NGF/proNGF (500 ng/mL) for 72 h. Once differentiation was completed, the cells were fixed in paraformaldehyde (PFA) 4% and incubated overnight with anti-MyHC (green) to evaluate the fusion index by immunofluorescence. DAPI (blue) was used to counterstain the nuclei. Fusion index was calculated as the percentage of nuclei incorporated into myotubes on the total nuclei present in each field. All the experiments consisted of at least three biological replicates. Scale bars: 100 μm. Values are expressed as the mean ± SD. Statistical analysis was performed by using the Student’s t test. * p

    Journal: Cells

    Article Title: ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells

    doi: 10.3390/cells9102232

    Figure Lengend Snippet: Effect of proNGF administration and anti-NGF/proNGF addition on SCDM differentiation. ( A ) SCDMs were allowed to differentiate into myotubes in the differentiation medium (DM), in presence or not of proNGF (100 ng/mL) for 72 h. Cells were observed with bright field microscopy and three images for every single experiment were captured. ( B ) SCDMs were treated with anti-NGF/proNGF (500 ng/mL) and induced to differentiate in DM for 72 h. Three images for each experimental group were captured under a brightfield microscope, and then analyzed to evaluate myotube length, myotube diameter and myotube number. ( C ) SCDMs were induced to differentiate in the presence of proNGF (100 ng/mL) or anti-NGF/proNGF (500 ng/mL) for 72 h. Once differentiation was completed, the cells were fixed in paraformaldehyde (PFA) 4% and incubated overnight with anti-MyHC (green) to evaluate the fusion index by immunofluorescence. DAPI (blue) was used to counterstain the nuclei. Fusion index was calculated as the percentage of nuclei incorporated into myotubes on the total nuclei present in each field. All the experiments consisted of at least three biological replicates. Scale bars: 100 μm. Values are expressed as the mean ± SD. Statistical analysis was performed by using the Student’s t test. * p

    Article Snippet: Complete differentiation was achieved after 72 h. The beginning and duration of the treatment with 500 ng/mL of NGF/proNGF neutralizing antibody (Alomone labs, Jerusalem, Israel; ALM-006), 100 ng/mL of recombinant mouse proNGF (Alomone labs, Jerusalem, Israel; N-250), and/or 100 nM of LM11A-31 (Sigma Aldrich, Milan, Italy; SML0664) is indicated in the figure legends.

    Techniques: Microscopy, Incubation, Immunofluorescence

    Effect of proNGF administration and anti-NGF/proNGF addition on SCDM proliferation. ( A ) SCDMs were cultured in a proliferation medium and treated with proNGF (100 ng/mL) up to 48 h. Cells were counted at both 24 and 48 h to evaluate cell growth. ( B ) SCDM cell growth was evaluated as in A, in both control cells and SCDMs treated with neutralizing anti-NGF/proNGF antibody (500 ng/mL) for 48 h. ( C ) EdU staining and the ratio of EdU + cells from proliferating SCDMs, kept in a proliferation medium in the presence or not of proNGF (100 ng/mL) or anti-NGF/proNGF (500 ng/mL) for 48 h. DAPI was used to counterstain the nuclei. Three images for each experimental group were captured under a fluorescence microscope, and then analyzed to evaluate the percentage of EdU + cells. All the experiments consist of at least three biological replicates. Scale bar: 50 μm. Values are expressed as the mean ± SD. For cell proliferation, statistical analysis was performed by using two-way ANOVA, whereas the ratio of EdU + cells was analyzed by one-way ANOVA followed by Tukey’s post hoc test.

    Journal: Cells

    Article Title: ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells

    doi: 10.3390/cells9102232

    Figure Lengend Snippet: Effect of proNGF administration and anti-NGF/proNGF addition on SCDM proliferation. ( A ) SCDMs were cultured in a proliferation medium and treated with proNGF (100 ng/mL) up to 48 h. Cells were counted at both 24 and 48 h to evaluate cell growth. ( B ) SCDM cell growth was evaluated as in A, in both control cells and SCDMs treated with neutralizing anti-NGF/proNGF antibody (500 ng/mL) for 48 h. ( C ) EdU staining and the ratio of EdU + cells from proliferating SCDMs, kept in a proliferation medium in the presence or not of proNGF (100 ng/mL) or anti-NGF/proNGF (500 ng/mL) for 48 h. DAPI was used to counterstain the nuclei. Three images for each experimental group were captured under a fluorescence microscope, and then analyzed to evaluate the percentage of EdU + cells. All the experiments consist of at least three biological replicates. Scale bar: 50 μm. Values are expressed as the mean ± SD. For cell proliferation, statistical analysis was performed by using two-way ANOVA, whereas the ratio of EdU + cells was analyzed by one-way ANOVA followed by Tukey’s post hoc test.

    Article Snippet: Complete differentiation was achieved after 72 h. The beginning and duration of the treatment with 500 ng/mL of NGF/proNGF neutralizing antibody (Alomone labs, Jerusalem, Israel; ALM-006), 100 ng/mL of recombinant mouse proNGF (Alomone labs, Jerusalem, Israel; N-250), and/or 100 nM of LM11A-31 (Sigma Aldrich, Milan, Italy; SML0664) is indicated in the figure legends.

    Techniques: Cell Culture, Staining, Fluorescence, Microscopy