CHIR-99021 (CT99021) HCl

Validation & Quality Control

Product Citations(5)

Nat Protoc, 2012, 8(1), 162-175

Proc Natl Acad Sci U S A, 2012, 109(27), E1848-1857

Carcinogenesis, 2012, 33(3), 529-537

J Biol Chem, 2012, 287(2), 893-904

BMC Dev Biol, 2012, 12, 21

Customer Reviews(5) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

CHIR-99021 (CT99021) HCl is available in the following compound libraries:

Cambridge Cancer Compound Library(96-well) Chemical Library (96-well) Inhibitor Library (96-well)

Products used with CHIR-99021 (CT99021) HCl in previous publications

Targets	Related Products	References	Purpose
GSK-3 inhibitors	SB214763	[1][2][3]	Positive Control. Compared to the GSK-3 inhibition potency of CHIR-99021, BIO, SB214763, Tideglusib and AR-A014418.
	CHIR-98014	[4][5][6]
	Tideglusib	[7][3][4]
PI3K inhibitors	LY294002	[8][9][6]	PI3K-mediated activation of Akt results in inhibition of Gsk-3. Many studies on GSK-3 signaling also use PI3K inhibitors.
	PI-103	[10][11]
	Wortmannin	[12][13][11]
MEK inhibitors	PD0325901	[14][15][16]	GSK-3 is critical in downstream signaling for proteins in the Ras/Raf/MEK-1 pathway.
MEK inhibitors	U0126-EtOH	[6][17][13]
CDK inhibitor	Roscovitine	[6][18][11]	Inhibition of CDK5 activity in axons leads to activation of GSK3 by protein phosphatase 1.

Product Information

Compare GSK-3 Inhibitors

Research Area
Research Area
Dr. Antonino Maria Sparta demonstrates a breakthrough in leukemia research by utilizing two inhibitors produced by Selleck Chemicals.

Cat.No.	Product Name	Solubility (25°C)
S2924	CHIR-99021 (CT99021) HCl	100 mg/mL	100 mg/mL	20 mg/mL
S1263	CHIR-99021 (CT99021)	100 mg/mL	100 mg/mL	20 mg/mL
S2745	CHIR-98014	<1 mg/mL	4 mg/mL	<1 mg/mL
S1075	SB 216763	<1 mg/mL	23 mg/mL	<1 mg/mL
S2823	Tideglusib	<1 mg/mL	1 mg/mL	<1 mg/mL
S1590	TWS119	<1 mg/mL	64 mg/mL	<1 mg/mL
S2729	SB 415286	<1 mg/mL	72 mg/mL	72 mg/mL
S2926	TDZD-8	<1 mg/mL	44.5 mg/mL	44.5 mg/mL

Product Description

Biological Activity Protocol Chemical Information Customer Reviews Product Citations Tech Support & FAQs

Biological Activity

Description	CHIR-99021 HCl (CT99021) is hydrochloride of CHIR-99021, which is a GSK-3α and GSK-3β inhibitor with IC50 of 10 nM and 6.7 nM, respectively.
Targets	GSK-3α	GSK-3β
IC50	10 nM	6.7 nM ^[1]
In vitro	CHIR-99021 shows greater than 500-fold selectivity for GSK-3 versus its closest homologs CDC2 and ERK2, as well as other protein kinases. Furthermore, CHIR-99021 shows only weak binding to a panel of 22 pharmacologically relevant receptors and little inhibitory activity against a panel of 23 nonkinase enzymes. CHIR-99021 induces the activation of glycogen synthase (GS) in insulin receptor-expressing CHO-IR cells with EC50 of 0.763 μM. ^[1] In addition to simulating the actions of insulin, inhibition of GSK-3 by CHIR-99021 (3 μM) increases free cytosolic β-catenin by 1.9-fold, mimicking the canonical Wnt signaling pathway in 3T3-L1 preadipocytes. During any of the first 3 days of differentiation, CHIR-99021 treatment inhibits the preadipocyte differentiation with IC50 of 0.3 μM by blocking induction of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ). ^[2] Unlike lithium chloride and AR-A014418, CHIR-99021 treatment does not reduce the viability of INS-1E cells even at high concentrations. Instead, CHIR-99021 robustly increases the rate of proliferation of INS-1E cells in a dose-dependent manner, and significantly inhibits INS-E cell death induced by high glucose and high palmitate in a concentration-dependent manner. CHIR-99021 promotes primary beta cell replication in isolated rat islets at concentrations as low as 1 μM, with 2-3 fold increase of cell replication at 5 μM of CHIR-99021 treatment. ^[3]
In vivo	Oral administration of CHIR-99021 at 30 mg/kg enhances glucose metabolism in a rodent model of type 2 diabetes, with a maximal plasma glucose reduction of nearly 150 mg/dl 3-4 hours after administration, while plasma insulin remains at or below control levels. Oral administration of CHIR-99021 at 16 or 48 mg/kg 1 hour before oral glucose challenges in ZDF rats significantly improves glucose tolerance with 14% and 33% reduction in plasma glucose at 16 mg/kg and 48 mg/kg, respectively, and the higher dose of CHIR-99021 also reduces hyperglycemia before the oral glucose challenge. ^[1] Administration of CHIR-99021 significantly augments hematopoietic repopulation in recipient mice transplanted with mouse or human hematopoietic stem cells (HSCs), suggesting that GSK-3 is a specific modulator of HSC activity. ^[4]
Clinical Trials
Features

Protocol(Only for Reference)

Kinase Assay:
^[1]

Cell-free kinase assays	Polypropylene 96-well plates are filled with 300 μL/well buffer (50 mM tris HCl, 10 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 1 mM NaF, 0.01% BSA, pH 7.5) containing 27 nM GSK-3α or 29 nM GSK-3β, and 0.5 μM biotin-CREB peptide substrate. Various concentrations of CHIR-99021 are added in 3.5 μL of DMSO, followed by 50 μL of ATP stock to yield a final concentration of 1 μM ATP in all cell-free assays. After incubation, triplicate 100-μL aliquots are transferred to Combiplate eight plates containing 100 μL/well 50 μM ATP and 20 mM EDTA. After 1 hour, the wells are rinsed five times with PBS, filled with 200 μL of scintillation fluid, sealed, left 30 minutes, and counted in a scintillation counter. All steps are performed at room temperature.

Cell-free kinase assays

Polypropylene 96-well plates are filled with 300 μL/well buffer (50 mM tris HCl, 10 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol, 25 mM β-glycerophosphate, 1 mM NaF, 0.01% BSA, pH 7.5) containing 27 nM GSK-3α or 29 nM GSK-3β, and 0.5 μM biotin-CREB peptide substrate. Various concentrations of CHIR-99021 are added in 3.5 μL of DMSO, followed by 50 μL of ATP stock to yield a final concentration of 1 μM ATP in all cell-free assays. After incubation, triplicate 100-μL aliquots are transferred to Combiplate eight plates containing 100 μL/well 50 μM ATP and 20 mM EDTA. After 1 hour, the wells are rinsed five times with PBS, filled with 200 μL of scintillation fluid, sealed, left 30 minutes, and counted in a scintillation counter. All steps are performed at room temperature.

Cell Assay:
^[3]

Cell lines	INS-1E
Concentrations	Dissolved in DMSO, final concentrations ~ 20 μM
Incubation Time	1, or 4 days
Method	Cells are maintained for 24 hours in starvation medium (culture medium with only 5 mM glucose, 1% fetal calf serum). Then cells are exposed to various concentrations of CHIR-99021 for 1, or 4 days. Cell number is measured by staining of cellular DNA with CyQuant dye, which becomes fluorescent when bound to DNA. Fluorescence is measured after 30 minutes of incubation using the FLUOstar Optima reader. Cell replication is determined by BrdUrd incorporation. BrdUrd labeling solution is added to the medium for the last 4 hours before the cells are fixed using FixDenat solution and incubated with monoclonal anti-BrdUrd-POD antibodies. After substrate solution is added to each well, the light emission is measured in a microplate luminometer using the Analyst HT detection system.

Animal Study:
^[1]

Animal Models	Female db/db mice or male ZDF rats with type 2 diabetes
Formulation	Formulated as solutions in 20 mM citrate-buffered 15% Captisol or as fine suspensions in 0.5% carboxymethylcellulose.
Dosages	~48 mg/kg
Administration	Orally

1

References

Chemical Information

Download CHIR-99021 (CT99021) HCl SDF

Molecular Weight (MW)	501.8
Formula	C₂₂H₁₈Cl₂N_8.HCl
CAS No.	252917-06-9 (free base)
Synonyms	N/A

Solubility (25°C) DMSO 100 mg/mL Water 100 mg/mL Ethanol 20 mg/mL
Storage	2 years -20°CPowder
	2 weeks4°Cin DMSO
	6 months-80°Cin DMSO

Chemical Name	6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile hydrochloride

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Research Area

Customer Reviews (5)

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Rating

Source

PLoS ONE, 7(10), e47312. CHIR-99021 (CT99021) HCl purchased from Selleck

Method

Westernblot

Cell Lines

WM793/1205LU

Concentrations

2 uM

Incubation Time

2 h

Results

Phoshorylation of β-catenin is decreased and total b-catenin level is slightly increased as a result of CT99021 2-hour treatment.

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Rating

Source

STEM CELLS, 2012, 30, 1863–1874. CHIR-99021 (CT99021) HCl purchased from Selleck

Method

beating foci assay

Cell Lines

ESCs

Concentrations

1/3uM

Incubation Time

5 d

Results

Similarly, inhibition of GSK3 by LiCl [48], by CHIR 99021 [7], or by AR A014418 (a GSK3 inhibitor, which lacks side effects as impairment of cyclin functions [49]), restored the differentiation potential of Gpc4-mutant ESCs to a similar extent.

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Rating

Source

Journal of Genetics and Genomics 39 (2012) 643e651. CHIR-99021 (CT99021) HCl purchased from Selleck

Method

Rat embryoid bodies (rEBs) formation

Cell Lines

rES cells

Concentrations

0.75 uM

Incubation Time

4 d

Results

For the first 2 days of rEBs formation, rES cells were cultured in CM in the presence of Rho kinase inhibitor, Y-27632 (10 umol/L) and 2i (PD0325901, 0.1 umol/L; CHIR99021, 0.75 umol/L; Selleck, USA). Thereafter, we transferred the regular rEBs into CM supplemented with 2i and without Y-27632 for another 2 days. On day 4, abundant highquality rEBs formed and then 2i was eliminated from the media.

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Rating

Source

CHIR-99021 (CT99021) HCl purchased from Selleck

Method

Western blot

Cell Lines

PANC1 cells

Concentrations

5 µM

Incubation Time

72 h

Results

prolonged inhibition of GSK3 expression or activity by CHIR99021 correlated with increased levels of cleaved PARP and caspase-7 as well as a 6-fold increase in caspase-3 activity.

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Rating

Source

CHIR-99021 (CT99021) HCl purchased from Selleck

Method

Western blot/qRT-PCR analysis

Cell Lines

Human lung tissue

Concentrations

0.5/10 μM

Incubation Time

72 h

Results

As presented in Figure 6 and B, Western blot analysis showed β-CATENIN expression in 15 W human lung explants decreased significantly from newly isolated to in vitro cultured for 72 h (0 μM). However, β-CATENIN expression nearly increased back to newly isolated levels in human lung explants at 15 W treated with 5 μM CHIR 99021 for 72 h, but decreased again after treatment of 10 μM CHIR 99021. Meanwhile, qRT-PCR analyses showed increased expression of transcription factors (LEF1 , TCF4) and target genes (CYCLIN D1 , MMP7) in human lung explants (15 W) exposed to 5 μM CHIR 99021 compared with tissues exposed to 0 μM CHIR 99021, the levels of which were also higher than in the 10 μM CHIR 99021 groups (Figure C).

Product Citations (5)

Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. [Lian X, et al. Nat Protoc 2012;8(1), 162-175]
PubMed: 23257984
Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling. [Lian X, et al. Proc Natl Acad Sci U S A 2012;109(27), E1848-1857]
PubMed: 22645348
Inhibition of glycogen synthase kinase-3 activity triggers an apoptotic response in pancreatic cancer cells through JNK-dependent mechanisms. [Marchand B, et al. Carcinogenesis 2012;33(3), 529-537]
PubMed: 22201186

Evidence for irreversible inhibition of glycogen synthase kinase-3β by tideglusib. [Domínguez JM, et al. J Biol Chem 2012;287(2), 893-904]
PubMed: 22102280
Expression of canonical WNT/β-CATENIN signaling components in the developing human lung. [Zhang M, et al. BMC Dev Biol 2012;12, 21]
PubMed: 22846383

Tech Support & FAQs

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3Monday–Friday 9:00 AM–5:00 PM (Central Time)

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Related GSK-3 Inhibitors

SB 216763
SB 216763 is a potent and selective GSK-3α inhibitor with IC50 of 34.3 nM.
CHIR-99021 (CT99021)

CHIR-99021 (CT99021) is a GSK-3α and GSK-3β inhibitor with IC50 of 10 nM and 6.7 nM, respectively.
TWS119
TWS119 is a GSK-3β inhibitor with IC50 of 30 nM.
SB 415286
SB 415286 is a potent GSK3α and GSK3β inhibitor with IC50 of 78 nM and ~78 nM, respectively.
CHIR-98014
CHIR-98014 is a potent and selective GSK-3 inhibitor for GSK-3ɑ and GSK-3β with IC50 of 0.65 nM and 0.58 nM, respectively.
Tideglusib
Tideglusib (NP031112, NP-12) is a potent non ATP-competitive inhibitor of GSK3 with IC50 of 60 nM.
TDZD-8
TDZD-8 is a non-ATP competitive GSK-3β inhibitor with IC50 of 2 μM.
LY2090314
LY2090314 is a potent GSK-3 inhibitor for GSK-3α and GSK-3β with IC50 of 1.5 nM and 0.9 nM, respectively.
BEZ235 (NVP-BEZ235)

BEZ235 (NVP-BEZ235) is a dual ATP-competitive PI3K and mTOR inhibitor of p110α, p110γ, p110δ and p110β with IC50 of 4 nM, 5 nM, 7 nM and 75 nM, respectively, and also inhibits ATR with IC50 of 21 nM.
Deforolimus (Ridaforolimus)

Deforolimus (Ridaforolimus, AP23573, MK-8669) is a selective mTOR inhibitor with IC50 of 0.2 nM.

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Product Categories

Validation & Quality Control

Product Citations(5)

Customer Reviews(5) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

CHIR-99021 (CT99021) HCl is available in the following compound libraries:

Products used with CHIR-99021 (CT99021) HCl in previous publications

Product Information

Compare GSK-3 Inhibitors

Research Area

Product Description

Biological Activity

Protocol(Only for Reference)

Kinase Assay:
^[1]

Cell Assay:
^[3]

Animal Study:
^[1]

References

Chemical Information

Research Area

Customer Reviews (5)

Product Citations (5)

Tech Support & FAQs

If you have any other enquiries, please leave a message.

Related GSK-3 Inhibitors

PI3K/Akt/mTOR Pathway Products

Recently Viewed Items

Cat.No.	Product Name	Solubility (25°C)
Cat.No.	Product Name	Water	DMSO	Ethanol
S2924	CHIR-99021 (CT99021) HCl	100 mg/mL	100 mg/mL	20 mg/mL
S1263	CHIR-99021 (CT99021)	100 mg/mL	100 mg/mL	20 mg/mL
S2745	CHIR-98014	<1 mg/mL	4 mg/mL	<1 mg/mL
S1075	SB 216763	<1 mg/mL	23 mg/mL	<1 mg/mL
S2823	Tideglusib	<1 mg/mL	1 mg/mL	<1 mg/mL
S1590	TWS119	<1 mg/mL	64 mg/mL	<1 mg/mL
S2729	SB 415286	<1 mg/mL	72 mg/mL	72 mg/mL
S2926	TDZD-8	<1 mg/mL	44.5 mg/mL	44.5 mg/mL

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Product Categories

CHIR-99021 (CT99021) HCl

Validation & Quality Control

Product Citations(5)

Customer Reviews(5) in vivo invitation

Quality Control & MSDS

Related Compound Libraries

CHIR-99021 (CT99021) HCl is available in the following compound libraries:

Products used with CHIR-99021 (CT99021) HCl in previous publications

Product Information

Compare GSK-3 Inhibitors

Research Area

Product Description

Biological Activity

Protocol(Only for Reference)

Kinase Assay: [1]

Cell Assay: [3]

Animal Study: [1]

References

Chemical Information

Research Area

Customer Reviews (5)

Product Citations (5)

Tech Support & FAQs

If you have any other enquiries, please leave a message.

Related GSK-3 Inhibitors

PI3K/Akt/mTOR Pathway Products

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Kinase Assay:
^[1]

Cell Assay:
^[3]

Animal Study:
^[1]