Journal: Nature Communications

Article Title: Macrophage-derived netrin-1 promotes abdominal aortic aneurysm formation by activating MMP3 in vascular smooth muscle cells

doi: 10.1038/s41467-018-07495-1

Figure Lengend Snippet: Netrin-1 mobilizes Ca 2+ and drives MMP3 activity in vascular smooth muscle cells via its receptor neogenin-1. a Calcium integration curves in VSMCs stimulation as indicated. Data are expressed as 340/380 nm excitation ratio. n = 4. * P < 0.05. b MMP3 activity in murine VSMCs stimulated with netrin-1 in the presence or absence of calcium chelator BAPTA (10 µM). n = 6–7 per group. ** P < 0.01. c NFATc3 (green) and actin (F-actin, red) immunofluorescence staining in mouse VSMCs (top panel) or human VSMCs (lower panel) stimulated with netrin-1 (2.5 µg/ml) with or without BAPTA. White arrowheads indicate nuclear location, n = 4. Scale bars 20 µm. d MMP3 activity in mouse VSMCs stimulated with netrin-1 in the presence or absence of NFATc3 inhibitor, VIVIT (1 µM). n = 6–7 per group. *** P < 0.001. e Immunofluorescence staining of neogenin-1 (NEO-1, red) and alpha-smooth muscle actin (SMA, green) in aortic sections of ApoE −/− mice. Colocalization is shown in merge. Nuclei stained with DAPI (blue). Scale bar 50 µm. f MMP3 activity in VSMCs stimulated with recombinant netrin-1 in the presence of control IgG, anti-NEO1, or anti-UNC5B neutralizing antibody (10 µg/ml). n = 6 per group. * P < 0.05, NS not significant. g MMP3 activity in VSMCs transfected with siRNA control, Neo1 or Unc5b and stimulated with recombinant netrin-1. n = 4–9 per group. * P < 0.05, ** P < 0.01, NS = not significant. h Calcium integration curves in VSMCs stimulated as indicated. Data are expressed as 340/380 nm excitation ratio. * P < 0.05. i NFATc3 and F-actin immunofluorescence staining of VSMCs stimulated with netrin-1, in the presence of control IgG or anti-NEO1 blocking antibody. White arrowheads indicate nuclear translocations. Scale bar 20 µm. j MMP3 enzymatic activity in VSMCs co-cultured with WTMø in the presence of control IgG or anti-NEO1 antibody. n = 4–8 per group. * P < 0.05. Two-way ANOVA was used to assess statistical significance in a and h . Unpaired, two-tailed t -test was used for statistical analysis in b , d , f , g , and j . Error bars represent s.e.m

Article Snippet: In some assays, neutralizing antibodies, goat anti-neogenin-1 antibody (AF1079-SP, R&D Systems), goat anti-Unc5b antibody (AF1006-SP, R&D Systems) or goat IgG control (AB-108-C, R&D Systems) were added 30 min prior to stimulations.

Techniques: Activity Assay, Immunofluorescence, Staining, Recombinant, Control, Transfection, Blocking Assay, Cell Culture, Two Tailed Test

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: ( A, B ) ChIP-seq profiles of the ( A ) Robo3 and ( B ) Unc5b loci for DNTTIP1 in WT, Dnttip1 KO1 and Elmsan1 KO1 mESCs and for HDAC1 in WT mESCs. Promoter regions used for manual ChIP experiments in ( C ) and ( E ) are highlighted by orange boxes. DNTTIP1 ChIP-seq peaks were determined based on the average of two replicates each from WT mESCs and based on the average of two replicates each from the Dnttip1 KO1 and Elmsan1 KO1 clone, respectively. HDAC1 ChIP-seq data were obtained from GSE55437. ( C ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Robo3 locus as highlighted in ( A ). IgG was used as a control antibody. ( D ) qRT-PCR for Robo3 mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh. ( E ) qPCR from manual ChIP experiments against DNTTIP1, ELMSAN1 and HDAC1 from WT, Dnttip1 KO1 and Elmsan1 KO1 NE targeting the promoter of the Unc5b locus as highlighted in ( B ). IgG was used as a control antibody. ( F ) qRT-PCR for Unc5b mRNA in WT, Dnttip1 KO1 and Elmsan1 KO1 NE after 12 days of differentiation. Expression was normalized to Gapdh .

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

Techniques: ChIP-sequencing, Control, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: ( A ) WB for signaling components of the SLIT3/ROBO3 and NTN1/DCC/UNC5B signaling axes from CM and total cell lysates of WT and Dnttip1 KO1 NE after 12 days of differentiation. To enrich SLIT3 and NTN1 from CM, IPs were performed with SLIT3 and NTN1 antibodies from CM of WT and Dnttip1 KO1 NE. Actin is the loading control for the total cell lysates. ( B ) Assay to rescue the neurite outgrowth defects in Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with preblocking of Dnttip1 KO1 NE with IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. MAP2 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. To facilitate analysis the neuronal cell body (blue) and its neurites were manually traced with ImageJ software and for each sample one traced neuron is displayed in the inlet. The white scale bar represents 50 µm. ( C, D ) Quantification of ( C ) neurite length and ( D ) the total number of neurites per neuron from the MAP2 IF staining in ( B ) using ImageJ. ( C ) Neurite length was divided into two categories of short neurites <50 µm (green box plots) and longer neurites ≥50 µm (white box plots). ( C, D ) The neurites of 200 neurons were assessed per sample. One-way ANOVA was performed throughout where ***, p≤0.001; and ns, p>0.05 is not significant.

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

Techniques: Control, Recombinant, Staining, Software

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: ( A ) Chamber assay to rescue the network formation defects of GNP-derived neurons that were supplemented with CM of Dnttip1 KO1 NE. CM of Dnttip1 KO1 NE was supplemented with the recombinant signaling ligands SLIT3 and/or NTN1 from day 7–12 without or with pre-blocking of GNP-derived neurons with control IgG or signaling receptor antibodies against ROBO3 and/or UNC5B. TUBB3 IF staining was performed after 12 days of differentiation. Nuclei were stained with DAPI. The white scale bar represents 100 µm. ( B ) Quantification of neuronal network formation from the TUBB3 IF staining in ( A ) using ImageJ. The percentage of formed neuronal networks within the total population of TUBB3-positive neurons is displayed. A neuronal network was scored when a closed local circuit was detected around an individual neuron. Neuronal network formation was assessed for 100 neurons per sample in triplicate. Unpaired t-test was performed throughout where ***, p≤0.01.

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

Techniques: Boyden Chamber Assay, Derivative Assay, Recombinant, Blocking Assay, Control, Staining

Journal: eLife

Article Title: The histone deacetylase complex MiDAC regulates a neurodevelopmental gene expression program to control neurite outgrowth

doi: 10.7554/eLife.57519

Figure Lengend Snippet: MiDAC directly binds to and deacetylates H4K20ac on regulatory elements of pro-neural genes such as those of the axon guidance ligands SLIT3 and NTN1 resulting in the activation of these genes. Conversely, MiDAC inhibits the gene expression of negative regulators of neurogenesis such as SPRY4 and ID1 by binding and removing H3K27ac from their promoters and enhancers. SLIT3 and NTN1, the downstream targets of MiDAC, bind to their cognate receptors ROBO3 and UNC5B respectively thereby activating the signaling cascade responsible for promoting neurite outgrowth.

Article Snippet: The rescued neurite outgrowth defects of Dnttip1 KO neurons via SLIT3, NTN1 or SLIT3/NTN1 supplementation were blocked by using antibodies directed against the extracellular domain of the axon guidance receptors ROBO3 (R&D Systems, AF3155), UNC5B (R&D Systems, MAB1006), a combination of ROBO3/UNC5B or IgG (Millipore, 12–370) as a negative control.

Techniques: Activation Assay, Gene Expression, Binding Assay

Journal: PLoS ONE

Article Title: The Uncoordinated-5 Homolog B (UNC5B) Receptor Increases Myocardial Ischemia-Reperfusion Injury

doi: 10.1371/journal.pone.0069477

Figure Lengend Snippet: A) Relative protein expression in murine brain (Br), intestine (In), liver (Li), heart (He) and kidney (Ki) by Western Blot analysis (n = 3; pooled samples). B) Gating of leukocytes subpopulations such as lymphocytes, monocytes and neutrophil granulocytes. C) Percentage of positive gated neutrophil granulocytes, leukocytes, monocytes and T- and B-cells (n = 5). Representative histograms of UNC5B expression (red) of CD45+ leukocytes and CD15+ neutrophils are shown D) Immunofluorescence UNC5B (green = Alexa 488) co-staining on neutrophil leukocytes (CD45 marked) and granulocytes (CD15 marked). Merged images show the localization of UNC5B on the neutrophils and leukocytes (n = 3 per group).

Article Snippet: In subsets of experiments PMNs or endothelium were pre-incubated with UNC5B antibody (R&D Systems) or IgG control.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS ONE

Article Title: The Uncoordinated-5 Homolog B (UNC5B) Receptor Increases Myocardial Ischemia-Reperfusion Injury

doi: 10.1371/journal.pone.0069477

Figure Lengend Snippet: A ) PMN transmigration studies were performed in transwells using fMLP as chemoattractant in the lower compartment. PMNs were pre-incubated with either anti-UNC5B antibody or IgG control prior to transmigration. Number of transmigrated PMNs was assessed by MPO measurement. B ) Passive flux of FITC Dextran across HMEC endothelium monolayers. Flux was detected by measurement of passed FITC Dextran in the basal compartment (n = 6 per condition; * P <0.05 as indicated).

Article Snippet: In subsets of experiments PMNs or endothelium were pre-incubated with UNC5B antibody (R&D Systems) or IgG control.

Techniques: Transmigration Assay, Incubation, Control

Journal: PLoS ONE

Article Title: The Uncoordinated-5 Homolog B (UNC5B) Receptor Increases Myocardial Ischemia-Reperfusion Injury

doi: 10.1371/journal.pone.0069477

Figure Lengend Snippet: A) Comparison of UNC5B protein expression in UNC5B +/− and WT animals in different organs (n = 5 per group). B) Comparison of UNC5B transcriptional mRNA levels of organs normalized to brain tissue of WT animals (n = 5 per group). C) Infarct size in UNC5B +/− mice compared with WT mice after 60 minutes of myocardial ischemia followed by 2 hours reperfusion. Calculated is the percentage of necrotic tissue to AAR. D) and E) Correlating serum troponin I and IL-6 levels of UNC5B +/− and WT mice (n = 6 per group; * P <0.05; ** P <0.01 as indicated) F) Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infarcted tissue).

Article Snippet: In subsets of experiments PMNs or endothelium were pre-incubated with UNC5B antibody (R&D Systems) or IgG control.

Techniques: Comparison, Expressing, Staining

Journal: PLoS ONE

Article Title: The Uncoordinated-5 Homolog B (UNC5B) Receptor Increases Myocardial Ischemia-Reperfusion Injury

doi: 10.1371/journal.pone.0069477

Figure Lengend Snippet: A) Infarct size of mice with UNC5B specific siRNA treatment compared to mice received non-targeting siRNA after 60 minutes of myocardial ischemia followed by 2 hours reperfusion. Calculated is the percentage of necrotic tissue to AAR. B) and C) Correlating serum Troponin I and IL-6 levels of these mice (n = 6 per group; * P <0.05; *** P <0.001 as indicated). D) Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted tissue) of the siRNA treated mice.

Article Snippet: In subsets of experiments PMNs or endothelium were pre-incubated with UNC5B antibody (R&D Systems) or IgG control.

Techniques: Staining

Journal: PLoS ONE

Article Title: The Uncoordinated-5 Homolog B (UNC5B) Receptor Increases Myocardial Ischemia-Reperfusion Injury

doi: 10.1371/journal.pone.0069477

Figure Lengend Snippet: A ) Infarct size after IR in WT mice receiving antibody against UNC5B (2.5 µg/mouse) iv. 30 min before onset of surgery. Controls were treated with corresponding IgG antibody. Calculated is the percentage of necrotic tissue to AAR. B) and C) Correlating serum Troponin I and IL-6 levels of these mice. (n = 6 per group; * P <0.05; *** P <0.001 as indicated). D) Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted tissue) of the antibody treated mice.

Article Snippet: In subsets of experiments PMNs or endothelium were pre-incubated with UNC5B antibody (R&D Systems) or IgG control.

Techniques: Staining

Journal: PLoS ONE

Article Title: The Uncoordinated-5 Homolog B (UNC5B) Receptor Increases Myocardial Ischemia-Reperfusion Injury

doi: 10.1371/journal.pone.0069477

Figure Lengend Snippet: A) Infarct size following IR in WT and UNC5B +/− mice after neutrophil granulocyte depletion and subsequent anti- UNC5B antibody adminsitration. Calculated is the percentage of necrotic tissue to AAR. B) and C) Correlating serum Troponin I and IL-6 levels of these mice. (n = 4 per group). D) Representative TTC stained heart slices of myocardial infarcts (blue/dark = retrograde Evans blue staining; red and white = AAR; white = infracted necrotic tissue.

Article Snippet: In subsets of experiments PMNs or endothelium were pre-incubated with UNC5B antibody (R&D Systems) or IgG control.

Techniques: Staining