| High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells
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Author:
Date:
2018-01-05
[Abstract] Here we describe two assays to measure dense core vesicle (DCV) exocytosis-mediated cargo secretion in neuroendocrine cells. To conduct siRNA screens for novel genes in regulated DCV exocytosis, we developed a plate reader-based secretion assay using DCV cargo, NPY-Venus, and an orthogonal 3H-serotonin secretion assay. The NPY-Venus secretion assay was successfully used for a high throughput siRNA screen, and the serotonin secretion assay was used to validate hits identified from the screen (Sorensen, 2017; Zhang et al., 2017).
[摘要] 在这里我们描述了两种测定方法来测量神经内分泌细胞中的致密核心囊泡（DCV）胞吐作用介导的货物分泌。 为了在受调控的DCV胞吐作用中对新基因进行siRNA筛选，我们开发了使用DCV货物，NPY-金星以及正交3H-血清素分泌测定的基于读板器的分泌测定法。 NPY-金星分泌测定成功地用于高通量siRNA筛选，并且使用血清素分泌测定来验证从筛选中鉴定的命中（Sorensen，2017; Zhang等人，2017）。
【背景】致密核心囊泡（DCV）胞吐作用介导内分泌和神经内分泌细胞分泌蛋白质，肽和小分子。当蛋白质和肽激素被内质网上的核糖体合成时，蛋白质和肽激素进入分泌途径，并在跨高尔基网络（TGN）（Borgonovo等人，2006; Bowman等， >等人，2009）。小分子物质，如5-羟色胺，直接被胞质单胺转运蛋白（VMATs）从细胞质直接吸收到DCV的内腔（Sudhof，2004）。在非刺激条件下，货物分子被储存在靠近质膜的DCV内。当细胞接受增加细胞质Ca ...
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| Clonal Culture of Mouse Liver Progenitor Cells
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Author:
Date:
2015-10-20
[Abstract] Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and cholangiocytes. For analyzing their differentiation potential, clonal culture has been used for LPCs isolated by a cell sorter. In addition, we can use the culture to assess functions of target genes on differentiation potential of LPCs. This protocol describes the process of cell isolation and colony assay to examine proliferative and differentiation potential of LPCs.
[摘要] 肝干/祖细胞（LPC）被定义为分化成肝细胞和胆管细胞的双能细胞。 为了分析其分化潜力，克隆培养已经用于通过细胞分选仪分离的LPC。 此外，我们可以使用文化来评估目标基因的功能分化潜力的LPCs。 该协议描述细胞分离和集落分析的过程，以检查增殖和分化潜力的LPCs。
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| [14C]-Tryptophan Metabolic Tracing in Liver Cancer Cells
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Author:
Date:
2015-09-05
[Abstract] Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for many NAD+-consuming proteins with diverse biological functions. Oscillations in NAD+ levels may influence several cellular signaling pathways. NAD+ synthesis via Preiss-Handler route (salvage reactions) has been extensively reported. However, the contribution of L-tryptophan/kynurenine catabolism in de novo NAD+ synthesis is poorly understood. Using L-[14C]-tryptophan tracing in four liver cancer cell lines and siRNA-mediated silencing of arylformamidase (AFMID), a key enzyme involved in L-tryptophan degradation, we demonstrate the contribution of L-tryptophan catabolism in de novo synthesis of NAD+ pools. NAD+ modulation is therefore important in ...
[摘要] 烟酰胺腺嘌呤二核苷酸（NAD + ）是许多具有不同生物功能的NAD +消耗蛋白的辅酶。 NAD + 水平的振荡可能影响几种细胞信号传导途径。 通过Preiss-Handler途径（补救反应）的NAD +合成已被广泛报道。 然而，对L-色氨酸/犬尿氨酸分解代谢在从头NAD +合成中的贡献知之甚少。 在四种肝癌细胞系中使用L- [14 C] - 色氨酸示踪和siRNA介导的芳基甲酰胺酶（AFMID）的沉默，其是参与L-色氨酸降解的关键酶，我们证明L-色氨酸分解代谢在从头合成中的贡献 NAD + 池。 因此，NAD + 调节在维持细胞稳态和根据营养物可用性的适当的细胞功能中是重要的。
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