CHIR-99021 (CT99021)
(Synonyms: CHIR99021, CHIR-99021, CHIR 99021, CT99021,GSK-3 Inhibitor XVI) 目录号 : GC16702
一种选择性的GSK3抑制剂
Cas No.:252917-06-9
Sample solution is provided at 25 µL, 10mM.
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| Cell experiment [1]: | |
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Cell lines |
Human Tenon's fibroblasts |
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Preparation Method |
Human Tenon's fibroblasts (HTFs) were pretreated with CHIR-99021, followed by treatment with 5 ng/mL of TGF-β for 30 minutes. For a quantitative evaluation of the level of gene transcription, quantitative real-time PCR was performed. |
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Reaction Conditions |
5, 10 µM CHIR-99021 for 48h. |
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Applications |
When HTFs were treated with 5 µM of CHIR 99021, with or without the addition of 5 ng/mL of TGF-b, there was a significant decrease in the production of the active form of GSK-3b, fibronectin, collagen Ia, and a-SMA.CHIR-99021 treatment attenuated the effects of TGF-b treatment, which had led to a significant increase in the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios. |
| Animal experiment [2]: | |
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Animal models |
C57BL/6J mice |
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Preparation Method |
The acquisition of operant alcohol and sucrose self-administration was established. Next, the selective GSK-3 inhibitor CHIR-99021 was injected 45 min prior to the start of the self-administration session. A maximum of 2 drug injections per week were conducted to ensure that responding returned to baseline after drug administration. |
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Dosage form |
CHIR-99021 0, 1, 3, or 10 mg/kg, i.p. injection |
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Applications |
CHIR-99021 (10 mg/kg) significantly increased the rate of alcohol-reinforced responding as compared to vehicle and that this effect emerged during and persisted throughout the second half of the 1-h session. The lower doses of CHIR 99021 did not alter alcohol reinforced response rate at any point throughout the session. |
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References: [1]. Lee SY, Chae MK, et al. The Effect of CHIR 99021, a Glycogen Synthase Kinase-3β Inhibitor, on Transforming Growth Factor β-Induced Tenon Fibrosis. Invest Ophthalmol Vis Sci. 2021 Dec 1;62(15):25. [2]. Faccidomo S, Holstein SE, et al. Pharmacological inhibition of glycogen synthase kinase 3 increases operant alcohol self-administration in a manner associated with altered pGSK-3β, protein interacting with C kinase and GluA2 protein expression in the reward pathway of male C57BL/6J mice. Behav Pharmacol. 2020 Feb;31(1):15-26. |
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CHIR-99021 is the most commonly used GSK-3β inhibitor and is considered the standard small-molecule Wnt agonist.CHIR-99021 is a potent inhibitor with high selectivity[[1].
CHIR-99021 led to a marked recovery in cell growth and viability suppressed by CDX2 overexpression. CHIR-99021 restored the protein levels of cyclin D1, c-myc, and β-catenin inhibited by overexpression of CDX2, as well as the cell growth and viability[2].
When the Human Tenon's fibroblasts(HTFs) were treated with TGF-β, a significant increase in the active form of GSK-3β was observed. A significant decrease in the active form of GSK-3β and molecules associated with fibrosis by TGF-β was noted in HTFs treated with CHIR-99021. CHIR-99021 treatment reduced the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios in HTFs and attenuated HTF migration[3].
The GSK-3 inhibitor CHIR 99021 trihydrochloride (0–10 mg/kg, ip) was injected 45-min prior to self-administration sessions in a counterbalanced design. After completion of the self-administration dose-effect curve, potential locomotor effects of the GSK-3 inhibitor were assessed. CHIR 99021 (10 mg/kg) dose-dependently increased alcohol reinforced responding with no effect on sucrose self-administration or locomotor activity. CHIR 99021 (10 mg/kg) significantly decreased pGSK-3β expression in all brain regions tested, reduced PICK1 and increased GluA2 total expression only in the NAcb.?Signaling through the GSK-3 / PICK1 / GluA2 molecular pathway drives the positive reinforcing effects of the drug, which are required for abuse liability[4].
CHIR-99021是最常用的GSK-3β抑制剂，被认为是标准的小分子Wnt激动剂。它是一种高选择性的强效抑制剂。
CHIR-99021可以显著促进细胞生长和存活，这些被CDX2过度表达所抑制的。CHIR-99021恢复了由于CDX2过度表达而受到抑制的cyclin D1、c-myc和β-catenin蛋白水平，同时也恢复了细胞生长和存活[2]。
当人类肌腱成纤维细胞（HTFs）接受TGF-β处理时，活性GSK-3β的显著增加。而在接受CHIR-99021处理的HTFs中，活性GSK-3β和与纤维化相关分子的活性形式明显降低。CHIR-99021治疗减少了HTFs中磷酸化Smad2/Smad2和磷酸化Smad3/Smad3比率，并减轻了HTF迁移[3]。
在一个平衡设计中，给小鼠注射GSK-3抑制剂CHIR 99021三盐酸盐（0-10毫克/千克，腹腔注射）45分钟后进行自我管理实验。完成自我管理剂量效应曲线后，评估了GSK-3抑制剂的潜在运动影响。CHIR 99021（10毫克/千克）依赖于剂量增加了对酒精的强化反应，在糖分自我管理或运动活动方面没有影响。 CHIR 99021（10毫克/千克）显着降低了所有测试大脑区域中pGSK-3β表达，并仅在NAcb中减少PICK1并增加GluA2总表达。通过GSK-3 / PICK1 / GluA2分子途径信号传导驱动药物的积极强化作用是滥用倾向所必需的[4]。
References:
[1].Law SM, Zheng JJ. Premise and peril of Wnt signaling activation through GSK-3β inhibition. iScience. 2022 Mar 25;25(4):104159.
[2].Yu J, Liu D, et al. CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling via transactivation of GSK-3β and Axin2 expression. Cell Death Dis. 2019 Jan 10;10(1):26.?
[3].Lee SY, Chae MK, et al. The Effect of CHIR 99021, a Glycogen Synthase Kinase-3β Inhibitor, on Transforming Growth Factor β-Induced Tenon Fibrosis. Invest Ophthalmol Vis Sci. 2021 Dec 1;62(15):25.
[4].Faccidomo S, Holstein SE, et al. Pharmacological inhibition of glycogen synthase kinase 3 increases operant alcohol self-administration in a manner associated with altered pGSK-3β, protein interacting with C kinase and GluA2 protein expression in the reward pathway of male C57BL/6J mice. Behav Pharmacol. 2020 Feb;31(1):15-26.?
| Cas No. | 252917-06-9 | SDF | |
| 别名 | CHIR99021, CHIR-99021, CHIR 99021, CT99021,GSK-3 Inhibitor XVI | ||
| 化学名 | 6-((2-((4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino)nicotinonitrile | ||
| Canonical SMILES | N#CC1=CC=C(NCCNC2=NC=C(C3=NC=C(C)N3)C(C4=CC=C(Cl)C=C4Cl)=N2)N=C1 | ||
| 分子式 | C22H18Cl2N8 | 分子量 | 465.34 |
| 溶解度 | ≥ 23.3mg/mL in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.149 mL | 10.7448 mL | 21.4897 mL |
| 5 mM | 0.4298 mL | 2.149 mL | 4.2979 mL |
| 10 mM | 0.2149 mL | 1.0745 mL | 2.149 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Generation of pig induced pluripotent stem cells using an extended pluripotent stem cell culture system
Background: Pigs have emerged as one of the most popular large animal models in biomedical research, which in many cases is considered as a superior choice over rodent models. In addition, transplantation studies using pig pluripotent stem (PS) cell derivatives may serve as a testbed for safety and efficacy prior to human trials. Recently, it has been shown that mouse and human PS cells cultured in LCDM (recombinant human LIF, CHIR 99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride) medium exhibited extended developmental potential (designated as extended pluripotent stem cells, or EPS cells), which could generate both embryonic and extraembryonic tissues in chimeric mouse conceptus. Whether stable pig induced pluripotent stem (iPS) cells can be generated in LCDM medium and their chimeric competency remains unknown. Methods: iPS cells were generated by infecting pig pericytes (PC) and embryonic fibroblasts (PEFs) with a retroviral vector encoding Oct4, Sox2, Klf4, and cMyc reprogramming factors and subsequently cultured in a modified LCDM medium. The pluripotency of PC-iPS and PEF-iPS cells was characterized by examining the expression of pluripotency-related transcription factors and surface markers, transcriptome analysis, and in vitro and in vivo differentiation capabilities. Chimeric contribution of PC-iPS cells to mouse and pig conceptus was also evaluated with fluorescence microscopy, flow cytometry, and PCR analysis. Results: In this study, using a modified version of the LCDM medium, we successfully generated iPS cells from both PCs and PEFs. Both PC-iPS and PEF-iPS cells maintained the stable "dome-shaped" morphology and genome stability after long-term culture. The immunocytochemistry analyses revealed that both PC-iPS and PEF-iPS cells expressed OCT4, SOX2, and SALL4, but only PC-iPS cells expressed NANOG and TRA-1-81 (faint). PC-iPS and PEF-iPS cells could be differentiated into cell derivatives of all three primary germ layers in vitro. The transcriptome analysis showed that PEF-iPS and PC-iPS cells clustered with pig ICM, Heatmap and volcano plot showed that there were 1475 differentially expressed genes (DEGs) between PC-iPS and PEF-iPS cells (adjusted p value < 0.1), and the numbers of upregulated genes and downregulated genes in PC-iPS cells were 755 and 720, respectively. Upregulated genes were enriched with GO terms including regulation of stem cell differentiation, proliferation, development, and maintenance. And KEGG pathway enrichment in upregulated genes revealed Wnt, Jak-STAT, TGF-β, P53, and MAPK stem cell signaling pathways. Fluorescence microscopy and genomic PCR analyses using pig mtDNA-specific and GFP primers showed that the PC-iPS cell derivatives could be detected in both mouse and pig pre-implantation blastocysts and post-implantation conceptuses. Quantitative analysis via flow cytometry revealed that the chimeric contribution of pig PC-iPS cells in mouse conceptus was up to 0.04%. Conclusions: Our findings demonstrate that stable iPS cells could be generated in LCDM medium, which could give rise to both embryonic and extraembryonic cells in vivo. However, the efficiency and level of chimeric contribution of pig LCDM-iPS cells were found low.
CHIR-99021 regulates mitochondrial remodelling via β-catenin signalling and miRNA expression during endodermal differentiation
Mitochondrial remodelling is a central feature of stem cell differentiation. However, little is known about the regulatory mechanisms during these processes. Previously, we found that a pharmacological inhibitor of glycogen synthase kinase-3α and -3β, CHIR-99021, initiates human adipose stem cell differentiation into human definitive endodermal progenitor cells (hEPCs), which were directed to differentiate synchronously into hepatocyte-like cells after further treatment with combinations of soluble factors. In this study, we show that CHIR-99021 promotes mitochondrial biogenesis, the expression of PGC-1α (also known as PPARGC1A), TFAM and NRF1 (also known as NFE2L1), oxidative phosphorylation capacities, and the production of reactive oxygen species in hEPCs. Blocking mitochondrial dynamics using siRNA targeting DRP1 (also known as DNM1L) impaired definitive endodermal differentiation. Downregulation of β-catenin (CTNNB1) expression weakened the effect of CHIR-99021 on the induction of mitochondrial remodelling and the expression of transcription factors for mitochondrial biogenesis. Moreover, CHIR-99021 decreased the expression of miR-19b-2-5p, miR-23a-3p, miR-23c, miR-130a-3p and miR-130a-5p in hEPCs, which target transcription factors for mitochondrial biogenesis. These data demonstrate that CHIR-99021 plays a role in mitochondrial structure and function remodelling via activation of the β-catenin signalling pathway and inhibits the expression of miRNAs during definitive endodermal differentiation.This article has an associated First Person interview with the first author of the paper.
In Ovo Injection of CHIR-99021 Promotes Feather Follicle Development via Modulating the Wnt Signaling Pathway and Transcriptome in Goose Embryos ( Anser cygnoides)
Feather performs important physiological functions in birds, and it is also one of the economic productions in goose farming. Understanding and modulating feather follicle development during embryogenesis are essential for bird biology and the poultry industry. CHIR-99021 is a potent Wnt/β-catenin signaling pathway activator associated with feather follicle development. In this study, goose embryos (Anser cygnoides) received an in ovo injection of CHIR-9902, which was conducted at the beginning of feather follicle development (E9). The results showed that feather growth and feather follicle development were promoted. The Wnt signaling pathway was activated by the inhibition of GSK-3β. Transcriptomic analyses showed that the transcription changes were related to translation, metabolism, energy transport, and stress in dorsal tissue of embryos that received CHIR-99021, which might be to adapt and coordinate the promoting effects of CHIR-99021 on feather follicle development. This study suggests that in ovo injection of CHIR-99021 is a potential strategy to improve feather follicle development and feather-related traits for goose farming and provides profiling of the Wnt signaling pathway and transcriptome in dorsal tissue of goose embryos for further understanding of feather follicle development.
CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling via transactivation of GSK-3β and Axin2 expression
Caudal-related homeobox transcription factor 2 (CDX2), an intestine-specific nuclear transcription factor, has been strongly implicated in the tumourigenesis of various human cancers. However, the functional role of CDX2 in the development and progression of colorectal cancer (CRC) is not well known. In this study, CDX2 knockdown in colon cancer cells promoted cell proliferation in vitro, accelerated tumor formation in vivo, and induced a cell cycle transition from G0/G1 to S phase, whereas CDX2 overexpression inhibited cell proliferation. TOP/FOP-Flash reporter assay showed that CDX2 knockdown or CDX2 overexpression significantly increased or decreased Wnt signaling activity. Western blot assay showed that downstream targets of Wnt signaling, including β-catenin, cyclin D1 and c-myc, were up-regulated or down-regulated in CDX2-knockdown or CDX2-overexpressing colon cancer cells. In addition, suppression of Wnt signaling by XAV-939 led to a marked suppression of the cell proliferation enhanced by CDX2 knockdown, whereas activation of this signaling by CHIR-99021 significantly enhanced the cell proliferation inhibited by CDX2 overexpression. Dual-luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays further confirmed that CDX2 transcriptionally activates glycogen synthase kinase-3β (GSK-3β) and axis inhibition protein 2 (Axin2) expression by directly binding to the promoter of GSK-3β and the upstream enhancer of Axin2. In conclusion, these results indicated that CDX2 inhibits the proliferation and tumor formation of colon cancer cells by suppressing Wnt/β-catenin signaling.
The ground state of embryonic stem cell self-renewal
In the three decades since pluripotent mouse embryonic stem (ES) cells were first described they have been derived and maintained by using various empirical combinations of feeder cells, conditioned media, cytokines, growth factors, hormones, fetal calf serum, and serum extracts. Consequently ES-cell self-renewal is generally considered to be dependent on multifactorial stimulation of dedicated transcriptional circuitries, pre-eminent among which is the activation of STAT3 by cytokines (ref. 8). Here we show, however, that extrinsic stimuli are dispensable for the derivation, propagation and pluripotency of ES cells. Self-renewal is enabled by the elimination of differentiation-inducing signalling from mitogen-activated protein kinase. Additional inhibition of glycogen synthase kinase 3 consolidates biosynthetic capacity and suppresses residual differentiation. Complete bypass of cytokine signalling is confirmed by isolating ES cells genetically devoid of STAT3. These findings reveal that ES cells have an innate programme for self-replication that does not require extrinsic instruction. This property may account for their latent tumorigenicity. The delineation of minimal requirements for self-renewal now provides a defined platform for the precise description and dissection of the pluripotent state.