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CHIR-99021 (CT99021)

Catalog No.: GC16702

CHIR-99021 (CT99021) (CHIR-99021) is a potent and selective GSK-3α/β inhibitor with IC50s of 10 nM and 6.7 nM. CHIR-99021 (CT99021) shows >500-fold selectivity for GSK-3 over CDC2, ERK2 and other protein kinases. CHIR-99021 (CT99021) is also a potent Wnt/β-catenin signaling pathway activator. CHIR-99021 (CT99021) enhances mouse and human embryonic stem cells self-renewal. CHIR-99021 (CT99021) induces autophagy.

CHIR-99021 (CT99021) Chemical Structure

Size Price Stock Qty
10mM (in 1mL DMSO)
$73.00
In stock
5mg
$50.00
In stock
10mg
$70.00
In stock
25mg
$151.00
In stock
100mg
$416.00
In stock

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Sample solution is provided at 25 µL, 10mM.

Product Documents

Quality Control & SDS

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Protocol

Cell experiment [1]:

Cell lines

Human Tenon's fibroblasts

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.

Reaction Conditions

5, 10 µM CHIR-99021 for 48h.

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]:

Animal models

C57BL/6J mice

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.

Dosage form

CHIR-99021 0, 1, 3, or 10 mg/kg, i.p. injection

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. 

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. 

Background

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].

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. 

Chemical Properties

Cas No. 252917-06-9 SDF
Synonyms CHIR99021, CHIR-99021, CHIR 99021, CT99021,GSK-3 Inhibitor XVI
Chemical Name 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
Formula C22H18Cl2N8 M.Wt 465.34
Solubility ≥ 23.3mg/mL in DMSO Storage Store at 4°C
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request

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Research Update

CHIR-99021 regulates mitochondrial remodelling via β-catenin signalling and miRNA expression during endodermal differentiation

J Cell Sci2019 Jul 31;132(15):jcs229948.PMID: 31289194DOI: 10.1242/jcs.229948

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.

The Effect of CHIR 99021, a Glycogen Synthase Kinase-3β Inhibitor, on Transforming Growth Factor β-Induced Tenon Fibrosis

Invest Ophthalmol Vis Sci2021 Dec 1;62(15):25.PMID: 34940783DOI: 10.1167/iovs.62.15.25

Purpose: This study investigated the effect of glycogen synthase kinase-3β (GSK-3β) inhibition on the fibrosis of human Tenon's fibroblasts (HTFs) induced by transforming growth factor-β (TGF-β).
Methods: Quantitative real-time PCR and Western blot analyses were performed to determine the expression levels of molecules associated with the fibrosis of HTFs by TGF-β (fibronectin, collagen Iα, and α-smooth muscle actin) and GSK-3β. The levels of phosphorylated Smad2 and Smad3 were also analyzed in the presence of the GSK-3β inhibitor CHIR 99021. The wound healing assay was performed to determine the effect of CHIR 99021 on the migration of HTFs. All experiments were conducted using primary cultured HTFs or human tenon tissues obtained from normal subjects and patients with glaucoma.
Results: Treatment with TGF-β resulted in an increase in the levels of molecules associated with the fibrosis of HTFs. The expression levels of these molecules were higher in the tenon tissues obtained from patients with glaucoma than those from normal subjects. When the HTFs were treated with TGF-β, a significant increase in the active form of GSK-3β (Y216) 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.
Conclusions: Our results demonstrated the effect of GSK-3β inhibition on the regulation of TGF-β-mediated fibrosis of HTFs, suggesting GSK-3β to be a potential target for maintaining bleb function after glaucoma filtration surgery.

GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration

PLoS One2021 May 5;16(5):e0251173.PMID: 33951093DOI: 10.1371/journal.pone.0251173

Cerebral organoids generated from human pluripotent stem cells (hiPSCs) are unique in their ability to recapitulate human-specific neurodevelopmental events. They are capable of modeling the human brain and its cell composition, including human-specific progenitor cell types; ordered laminar compartments; and both cell-specific transcriptional signatures and the broader telencephalic transcriptional landscape. The serine/threonine kinase, GSK3β, plays a critical role in neurodevelopment, controlling processes as varied as neurogenesis, morphological changes, polarization, and migration. In the generation of cerebral organoids, inhibition of GSK3β at low doses has been used to increase organoid size and decrease necrotic core. However, little is known of the effects of GSK3β inhibition on organoid development. Here, we demonstrate that while low dose of GSK3β inhibitor CHIR 99021 increases organoid size, higher dose actually reduces organoid size; with the highest dose arresting organoid growth. To examine the mechanisms that may contribute to the phenotypic size differences observed in these treatment groups, we show that low dose of CHIR 99021 increases cell survival, neural progenitor cell proliferation and neuronal migration. A higher dose, however, decreases not only apoptosis but also proliferation, and arrests neural differentiation, enriching the pool of neuroepithelial cells, and decreasing the pools of early neuronal progenitors and neurons. These results reveal new mechanisms of the pleiotropic effects of GSK3β during organoid development, providing essential information for the improvement of organoid production and ultimately shedding light on the mechanisms of embryonic brain development.

Glycogen synthase kinase 3β inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells

Cytotherapy2020 Feb;22(2):91-105.PMID: 31980369DOI: 10.1016/j.jcyt.2019.12.007

Aim: Mesenchymal stem cells (MSCs) are immunomodulatory, non-teratogenic and multipotent alternatives to embryonic or induced pluripotent stem cells (ESCs or iPSCs). However, the potency of MSCs is not equivalent to the pluripotency of ESCs or iPSCs. We used CHIR 99021 to improve current protocols and methods of differentiation for the enhanced transdifferentiation potency of MSCs.
Main methods: We used Flurescence activated cell sorter (FACS) for MSC immunophenotyping and biochemical assay for demonstrating the trilineage potential of MSCs. We used real-time polymerase chain reaction, immunocytochemistry and Western blotting assay for analyzing the expression of lineage-specific markers.
Key findings: CHIR 99021 treatment of MSCs resulted in enhanced transdifferentiation into neurological, hepatogenic and cardiomyocyte lineages with standardized protocols of differentiation. CHIR 99021-treated MSCs showed increased nuclear localization of β-catenin. These MSCs showed a significantly increased deposition of active histone marks (H3K4Me3, H3K36Me3), whereas no change was observed in repressive marks (H3K9Me3, H3K27Me3). Differential methylation profiling showed demethylation of the transcription factor OCT4 promoter region with subsequent analysis revealing increased gene expression and protein content. The HLA-DR antigen was absent in CHIR 99021-treated MSCs and their differentiated cell types, indicating their immune-privileged status. Karyotyping analysis showed that CHIR 99021-treated MSCs were genomically stable. Teratoma analysis of nude mice injected with CHIR 99021-treated MSCs showed the increased presence of cell types of mesodermal origin at the site of injection.
Significance: MSCs pretreated with CHIR 99021 can be potent, abundant alternative sources of stem cells with enhanced differentiation capabilities that are well suited to cell-based regenerative therapy.

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