CME/clathrin-mediated endocytosis

Pitstop 2（20–40 μM；30 分钟）可防止 J774A.1 巨噬细胞内吞转铁蛋白，同时保持细胞活力[1]。 Pitstop 2（20–40 μM；30 分钟）对 J774A.1 巨噬细胞内化霍乱毒素 B 的能力没有影响[1]。 Pitstop 2（0.001-100 μM；6 小时）抑制 HeLa 细胞的纺锤体，阻止其进行有丝分裂[2]。 Pitstop 2（1-30 μM；24 小时）抑制分裂癌细胞的生长并导致细胞凋亡[2]。 Pitstop 2（1-30 μM；48 小时）对非致瘤性 NIH3T3 成纤维细胞的增殖和保持活力的能力没有影响[2]。

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杜氏利什曼原虫是一种细胞内原生动物寄生虫，可引起内脏利什曼病，这是全球死亡和发病的主要原因。宿主质膜是利什曼原虫进入细胞内部的入口。尽管已经证明几种宿主细胞膜受体参与杜氏利什曼原虫进入宿主细胞，但参与寄生虫内化的内吞途径尚不清楚。在这项工作中，我们探索了杜氏利什曼原虫进入宿主巨噬细胞的内吞途径，利用针对两种主要内化途径的特异性抑制剂，即网格蛋白和小窝蛋白介导的内吞作用。我们发现，网格蛋白介导的内吞作用抑制剂pitstop 2不会影响杜氏利什曼原虫前鞭毛体进入宿主巨噬细胞。有趣的是，用金雀异黄素（一种小窝蛋白介导的内吞作用抑制剂）治疗后，观察到内化显著减少。宿主巨噬细胞内无鞭毛体负载的类似趋势支持了这些结果。这些结果表明，杜氏利什曼原虫利用小窝蛋白介导的内吞作用内化到宿主细胞中。我们的研究结果为杜氏利什曼原虫吞噬宿主细胞的机制提供了新的见解，并为开发针对利什曼原虫感染的新疗法提供了相关性。[1]

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背景：在中期，网格蛋白稳定有丝分裂纺锤体动粒（K）纤维。许多抗有丝分裂化合物靶向微管动力学。Pitstop 2™是网格蛋白末端结构域的第一个小分子抑制剂，可抑制网格蛋白介导的内吞作用。我们研究了它对网格蛋白在有丝分裂中的第二个功能的影响。
结果：Pitstop 2不会损害网格蛋白对纺锤体的募集，但一旦驻扎在纺锤体上，就会破坏其功能。Pitstop 2通过有丝分裂纺锤体完整性的丧失和纺锤体组装检查点的激活、酚复制网格蛋白的耗竭和极光A激酶的抑制，将HeLa细胞困在中期。
结论：Pitstop 2是研究网格蛋白纺锤体动力学的新工具。Pitstop 2降低了分裂HeLa细胞的存活率，但不影响分裂的非癌NIH3T3细胞，这表明网格蛋白可能是一种新的抗有丝分裂药物靶点。[2]

细胞活力测定[2]
细胞类型： HeLa 细胞
测试浓度： 1、3、10、30 μM
孵育时间：24小时
实验结果：以剂量依赖性方式显着减少活HeLa细胞总数。

[1]. Leishmania donovani Internalizes into Host Cells via Caveolin-mediated Endocytosis. Sci Rep. 2019 Sep 2; 9(1): 12636.

[2]. Inhibition of clathrin by pitstop 2 activates the spindle assembly checkpoint and induces cell death in dividing HeLa cancer cells. Mol Cancer. 2013 Jan 17; 12:4.

Leishmania donovani is an intracellular protozoan parasite that causes visceral leishmaniasis, a major cause of mortality and morbidity worldwide. The host plasma membrane serves as the portal of entry for Leishmania to gain access to the cellular interior. Although several host cell membrane receptors have been shown to be involved in the entry of Leishmania donovani into host cells, the endocytic pathway involved in the internalization of the parasite is not known. In this work, we explored the endocytic pathway involved in the entry of Leishmania donovani into host macrophages, utilizing specific inhibitors against two major pathways of internalization, i.e., clathrin- and caveolin-mediated endocytosis. We show that pitstop 2, an inhibitor for clathrin-mediated endocytosis, does not affect the entry of Leishmania donovani promastigotes into host macrophages. Interestingly, a significant reduction in internalization was observed upon treatment with genistein, an inhibitor for caveolin-mediated endocytosis. These results are supported by a similar trend in intracellular amastigote load within host macrophages. These results suggest that Leishmania donovani utilizes caveolin-mediated endocytosis to internalize into host cells. Our results provide novel insight into the mechanism of phagocytosis of Leishmania donovani into host cells and assume relevance in the development of novel therapeutics against leishmanial infection.[1]

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Background: During metaphase clathrin stabilises the mitotic spindle kinetochore (K)-fibres. Many anti-mitotic compounds target microtubule dynamics. Pitstop 2™ is the first small molecule inhibitor of clathrin terminal domain and inhibits clathrin-mediated endocytosis. We investigated its effects on a second function for clathrin in mitosis.
Results: Pitstop 2 did not impair clathrin recruitment to the spindle but disrupted its function once stationed there. Pitstop 2 trapped HeLa cells in metaphase through loss of mitotic spindle integrity and activation of the spindle assembly checkpoint, phenocopying clathrin depletion and aurora A kinase inhibition.
Conclusions: Pitstop 2 is therefore a new tool for investigating clathrin spindle dynamics. Pitstop 2 reduced viability in dividing HeLa cells, without affecting dividing non-cancerous NIH3T3 cells, suggesting that clathrin is a possible novel anti-mitotic drug target.[2]

C20H13BRN2O3S2

473.362821340561

471.955

1419320-73-2

Clathrin-IN-1;1332879-52-3

136246422

Yellow to brown solid powder

5.5

109

1

5

3

28

767

0

C1(C=CC=C2C=CC=CC=12)S(=O)(=O)NC1S/C(=C\C2C=CC(Br)=CC=2)/C(=O)N=1

CGDLWHGPJPVPDU-ATVHPVEESA-N

InChI=1S/C20H13BrN2O3S2/c21-15-10-8-13(9-11-15)12-17-19(24)22-20(27-17)23-28(25,26)18-7-3-5-14-4-1-2-6-16(14)18/h1-12H,(H,22,23,24)/b17-12-

(NZ)-N-[(5Z)-5-[(4-bromophenyl)methylidene]-4-oxo-1,3-thiazolidin-2-ylidene]naphthalene-1-sulfonamide

Pitstop 2; Clathrin-IN-1; (NZ)-N-[(5Z)-5-[(4-bromophenyl)methylidene]-4-oxo-1,3-thiazolidin-2-ylidene]naphthalene-1-sulfonamide; 1419093-54-1; (Z)-N-(5-(4-Bromobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl)naphthalene-1-sulfonamide; 1419320-73-2; 4g55; 1332879-52-3;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 62.5 mg/mL (132.03 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.28 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。