Supplementary Figure 3 from The Chemokine (CCL2–CCR2) Signaling Axis Mediates Perineural Invasion
Shizhi HeShuangba HeChun‐Hao ChenSylvie DebordeRichard L. BakstNatalya ChernichenkoWilliam F. McNamaraSei‐Young LeeFernando BarajasZhenkun YuHikmat Al‐AhmadieRichard J. Wong
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<p>A. A schematic of the DRG and cancer cell co-culture model depicts that cancer cells initially attach to DRG neurites. Over time, the cancer cells migrate along the neurites towards the DRG and also proliferate. B. Close up views of the photomicrographs from Figure 3 show an association between the shControl PC-3 cells and the DRG neurites (Figure 3A), but fewer associations between the shCCR-2 PC-3 cells and DRG neurites (Figure 3B).</p>Keywords:
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A combined assay to measure neurite outgrowth and B-50/GAP-43 levels in PC12 cells is reported. During NGF-induced neuritogenesis, B-50/GAP-43 expression was monitored by enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose-response and time curve of beta-NGF-induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of beta-NGF greater than or equal to 5 ng/ml and was accompanied by a linear increase in the amount of B-50/GAP-43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.
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Objective To investigate the effect of α-Synuclein(α-Syn) on the neurite outgrowth of brain neurons.Methods Neurons isolated from the neocortex of newborn rats were cultured and α-Syn was added into the culture medium to observe the effect of the protein on the neurite outgrowth of the neurons.In addition,the effect of different concentrations of α-Syn on the neurite outgrowth was studied.Results The mean neurite length of the neurons treated with α-Syn was significantly longer than that of control neurons at 1 h,2 h and 4 h after adding α-Syn.The effect of α-Syn on neurite outgrowth was enhanced with the increase of α-Syn concentration and was reversed by anti-α-Syn monoclonal antibody.Conclusion α-Syn can promote neurite outgrowth of primary rat brain neurons in a dose-dependent manner.
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Previous studies have shown that thrombin prevents neurite outgrowth and induces neurite retraction in some but not all neuroblastoma lines. In the present study, cells were induced to elaborate neurites by serum deprivation. Alternate cultures were treated to induce alterations in those kinase activities known to induce neurite outgrowth and to enhance deposition into neurites of stabilizing cytoskeletal consituents such as phosphorylated neurofilaments. The relative influence of thrombin on neurites generated under these various conditions was examined. Quantitation of neurite length as a function of somal diameter revealed shortening, but not complete retraction, of neurites under all conditions. However, the relative percentage of neurites that underwent thrombin-induced shortening was substantially reduced in cultures deprived of serum in combination with alteration of kinase activities. Neurite reduction, rather than complete elimination, indicates that the influence of thrombin on neurites may be relatively confined to the distal end of elaborating neurites, and that the deposition of certain cytoskeletal consituents may progressively lessen the requirement of developing neurites on (thrombin-mediated) surface protease inhibition. These findings further suggest that, even during relatively early stages in neuritogenesis, thrombin-mediated surface protease activity exerts a greater influence on neurite initiation and elongation rather than maintenance of existing neurites.
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Neurite outgrowth in culture provides an easy way to determine the effects of a particular substrate or exogenous factor on neuron behavior. Dissociated neurons can be plated on a variety of substrates and the length of the longest neurite outgrowth can be compared. Here, we describe how to isolate and dissociate dorsal root ganglion (DRG) neurons, culture them on coverslips, and measure longest neurite outgrowth.
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Identification and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function. In this study, we applied 10 different phospholipids to cultured cortical neurons and found that lysophosphatidylethanolamines (LPEs) with different fatty acid lengths, palmitoyl LPE (16:0 LPE) and stearoyl LPE (18:0 LPE), stimulate neurite growth in cultured cortical neurons. Noteworthy, inhibitor of Gq/11 protein inhibited 16:0 LPE-stimulated neurite outgrowth but not 18:0 LPE-stimulated neurite outgrowth. In contrast, inhibitor of Gi/Go proteins inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of PKC inhibitors on neurite outgrowth were different between 16:0 LPE- and 18:0 LPE-treated cultures. We also found that both 16:0 LPE and 18:0 LPE activate MAPK to the same extent. MAPK inhibitor completely inhibited 18:0 LPE-stimulated neurite outgrowth and partially inhibited 16:0 LPE-stimulated neurite outgrowth. These results suggest that 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through distinct signaling cascades in cultured cortical neurons and that distinct G protein-coupled receptors are involved in these processes.
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