CHIR-99021

Biological Activity

CHIR-99021 (CT99021) inhibits glycogen synthase kinase-3β (GSK3β, IC50 = 6.7 nm) and GSK3α (IC50 = 10 nm), inhibits preadipocyte differentiation (IC50 = 0.3 µM), possibly by blocking induction of C/EBPα and PPARγ, and mimics Wnt signaling in 3T3-L1 preadipocytes. CHIR99021 activates glycogen synthase in CHO-IR cells with EC50 of 763 nM. CHIR99201 does not exhibit cross-reactivity against cyclin-dependent kinases (CDKs) with 350-fold selectivity toward GSK-3β compared to CDKs.

Product Citations

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  Chem Biol Interact. 2023 Dec 1;386:110777.

  Assessment of doxorubicin toxicity using human cardiac organoids: A novel model for evaluating drug cardiotoxicity
  CHIR-99021 purchased from AbMole

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  Nature. 2022 Jul;607(7917):149-155.

  Deciphering the immunopeptidome in vivo reveals new tumour antigens
  CHIR-99021 purchased from AbMole

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  Mol Biol Cell. 2022 Jul 1;33(8):mbcP22031008.

  Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models
  CHIR-99021 purchased from AbMole

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  Acta Histochem. 2022 Aug;124(6):151931.

  Fibroblast-derived exosomal miRNA-133 promotes cardiomyocyte-like differentiation
  CHIR-99021 purchased from AbMole

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  Cancer Cell. 2021 Oct 11;39(10):1342-1360.e14.

  The CD155/TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer
  CHIR-99021 purchased from AbMole

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  Adv Healthc Mater. 2021 Aug;10(16):e2100821.

  Extracellular Matrix Stiffness Regulates DNA Methylation by PKCα-Dependent Nuclear Transport of DNMT3L
  CHIR-99021 purchased from AbMole

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  bioRxiv. 2021 Jul 1;450516.

  Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models
  CHIR-99021 purchased from AbMole

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  2020 Sep.

  Developing a model to assess the contribution of cytokeratin 19-expressing cells during multipotent stromal cell-induced islet regeneration
  CHIR-99021 purchased from AbMole

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  Stem Cells. 2019 Apr;37(4):516-528.

  Human Multipotent Stromal Cell Secreted Effectors Accelerate Islet Regeneration.
  CHIR-99021 purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Sci Rep (2015). Figure 3. CHIR-99021
	Method	qPCR
	Cell Lines	J1 mESCs and F9 mEC cells
	Concentrations	3 μM
	Incubation Time	24 h
	Results	By contrast, all of the ESCC miRNAs were downregulated by CHIR (Fig. 3a–e). The miR-200b-429 and miR-183-182 clusters, which are highly expressed in pluripotent cells, were downregulated by both BIO and CHIR (Fig. 3f, 3g).

	Source	Sci Rep (2015). Figure 1. CHIR-99021
	Method	immunofluorescence staining
	Cell Lines	J1 mESCs and F9 mEC cells
	Concentrations	3 μM
	Incubation Time	24 h
	Results	The relativemRNA expression level of Nanog was determined in response to BIO or CHIR in J1mESCs and F9mEC cells by qPCR. As expected, transcription of Nanog in J1 mESCs and F9 mEC cells was upregulated by both BIO and CHIR

Protocol (for reference only)

Cell Experiment
Cell lines	J1 mESCs or F9 mEC cells
Preparation method	Immunofluorescence staining The expression and subcellular localisation of β-catenin in J1 mESCs treated with 1 μM BIO or 3 μM CHIR for 24 h was detected by immunofluorescence staining. Nuclei were stained with DAPI. J1 mESCs or F9 mEC cells were fixed and permeabilized using immunostaining fixation buffer and then blocked in blocking buffer (Beyotime Institute of Biotechnology). Subsequently, cells were incubated with the indicated primary antibody overnight at 4°C, followed by three washes with washing buffer (Beyotime Institute of Biotechnology) for 5 min and then incubation with an Alexa Fluor 555-conjugated secondary antibody for 2 h at room temperature. Nuclei were stained with DAPI. Cells were photographed under an inverted fluorescence microscope (Nikon, Tokyo, Japan).
Concentrations	3µM
Incubation time	24h

Animal Experiment
Animal models	Akita type 1 diabetic mice and wild-type mice
Formulation	DMSO
Dosages	50 mg/kg, daily
Administration	Intraperitoneal injection

Chemical Information

Molecular Weight	465.34
Formula	C22H18Cl2N8
CAS Number	252917-06-9
Solubility (25°C)	DMSO 16 mg/mL
Storage	Powder          -20°C   3 years ;  4°C   2 years In solvent       -80°C   6 months ;  -20°C   1 month

Conversion of different model animals based on BSA (PMID: 27057123)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of Compound A used for a mouse (20 mg/kg) to a dose based on the BSA for a rat, multiply 20 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for Compound A of 10 mg/kg.

References

[1] Ye, et al. PLoS One. Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains.