Name: GSK-J4 Hydrochloride
Brand: Selleck Chemicals
SKU: S7070
Rating: 5 (64 reviews)

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Quality Control

Batch: Purity: 99.48%

99.48

Related Targets	HDAC JAK BET Histone Methyltransferase PKC PARP HIF PRMT EZH2 AMPK
Other Histone Demethylase Inhibitors	SP2509 GSK-LSD1 2HCl Ladademstat (ORY-1001) Dihydrochloride JIB-04 CPI-455 HCl OG-L002 IOX1 GSK J1 ML324 CPI-455

Cell Culture, Treatment & Working Concentration

Cell Lines	Assay Type	Concentration	Incubation Time	Formulation	Activity Description	PMID
CUTLL1	Growth inhibitory assay	2 μM		DMSO	affects cell growth	25132549
CUTLL1	Apoptosis assay	2 μM		DMSO	induces apoptosis	25132549
CUTLL1	Function assay	2 μM		DMSO	induces cell cycle arrest	25132549
CUTLL1	Kinase assay	6 μM		DMSO	leads to increased H3K27me3	25132549
SF7761	Kinase assay	6 μM		DMSO	increases K27 methylation	25401693
SF8628	Kinase assay	6 μM		DMSO	increases K28 methylation	25401693
H3.3	Kinase assay	6 μM		DMSO	increases K29 methylation	25401693
SF9012	Kinase assay	6 μM		DMSO	increases K30 methylation	25401693
SF9402	Kinase assay	6 μM		DMSO	increases K31 methylation	25401693
SF9427	Kinase assay	6 μM		DMSO	increases K32 methylation	25401693
human astrocytes	Kinase assay	6 μM		DMSO	increases K33 methylation	25401693
SF7761	Growth inhibitory assay	6 μM		DMSO	inhibits K27M glioma cell growth	25401693
SF8628	Growth inhibitory assay	6 μM		DMSO	inhibits K28M glioma cell growth	25401693
H3.3	Growth inhibitory assay	6 μM		DMSO	inhibits K29M glioma cell growth	25401693
SF9012	Growth inhibitory assay	6 μM		DMSO	inhibits K30M glioma cell growth	25401693
SF9402	Growth inhibitory assay	6 μM		DMSO	inhibits K31M glioma cell growth	25401693
SF9427	Growth inhibitory assay	6 μM		DMSO	inhibits K32M glioma cell growth	25401693
human astrocytes	Growth inhibitory assay	6 μM		DMSO	inhibits K33M glioma cell growth	25401693
TG neurons	Function assay	50 μM		DMSO	inhibits HSV-1 reactivation from sensory neurons	25552720
Th17	Function assay	80 nM		DMSO	inhibits cell differentiation	25840993
β-cells	Function assay	20 μM		DMSO	blunts IFNγ, Il-1β, and TNFα-induced chemokine gene expression	26505193
β-cells	Function assay	20 μM		DMSO	induces β-cell dysfunction	26505193
ESCs	Function assay	1.8 µM		DMSO	induces DNA damage along with activation of the DNA damage response	26759175
Raw 264.7	Function assay	0.8192 µM		DMSO	inhibits TNF-α production	26776360
MCF7	Function assay	1 to 10 uM	30 hrs		Inhibition of KDM5A in human MCF7 cells assessed as effect on H3K4me3 methylation levels at 1 to 10 uM after 30 hrs by Western blot analysis	30392349
MCF7	Function assay	1 to 10 uM	30 hrs		Inhibition of KDM5A in human MCF7 cells assessed as effect on H3K27me3 methylation levels at 1 to 10 uM after 30 hrs by Western blot analysis	30392349
RAW264.7		0.82 uM	24 hrs		Inhibition of LPS-induced TNFalpha production in mouse RAW264.7 cells 0.82 uM after 24 hrs by ELISA	26776360
RAW264.7			24 hrs		Inhibition of LPS-induced TNFalpha production in mouse RAW264.7 cells after 24 hrs by ELISA	26776360
Click to View More Cell Line Experimental Data

Chemical Information, Storage & Stability

Molecular Weight	453.96	Formula	C₂₄H₂₇N₅O₂.HCl	Storage (From the date of receipt)	3 years-20°Cpowder
CAS No.	1797983-09-5	Download SDF		Storage of Stock Solutions	1 year-80°Cin solvent 1 month-20°Cin solvent

Solubility

In vitro
Batch:

DMSO : 91 mg/mL (200.45 mM)
(Moisture-contaminated DMSO may reduce solubility. Use fresh, anhydrous DMSO.)

Ethanol : 91 mg/mL

Water : 10 mg/mL

Molarity Calculator

Mass	Concentration	Volume	Molecular Weight
=	×	×

Dilution Calculator Molecular Weight Calculator

In vivo Batch:
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 40%PEG300 2 5%Tween80 3 50%ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	5.000mg/ml (11.01mM)	Taking the 1 mL working solution as an example, add 50 μL of 100 mg/ml clarified DMSO stock solution to 400 μL of PEG300, mix evenly to clarify it; add 50 μL of Tween80 to the above system, mix evenly to clarify; then continue to add 500 μL of ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Homogeneous suspension	CMC-NA	≥5mg/ml	Taking the 1 mL working solution as an example, add 5 mg of this product to 1 ml of CMC-Na solution, mix evenly to obtain a homogeneous suspension with a final concentration of 5 mg/ml.
Clear solution	5%DMSO 1 20%PEG300 2 5%Tween80 3 70%ddH2O Validated by Selleck labs. Should you need adjustments to this formulation, contact our sales team for custom testing.	1.000mg/ml (2.20mM)	Taking the 1 mL working solution as an example, add 50 μL 20 mg/ml clarified DMSO stock solution to 200 μL PEG300, mix evenly to clarify it; add 50 μL Tween80 to the above system, mix evenly to clarify it; then continue to add 700 μL ddH2O to adjust the volume to 1 mL. The mixed solution should be used immediately for optimal results.

In vivo Formulation Calculator (Clear solution)

Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)

Dosage: mg/kg Average weight of animals: g Dosing volume per animal:μL Number of animals:

Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

Calculation results:

Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH₂O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.

Mechanism of Action

Targets/IC₅₀/Ki	JMJD3 (Cell-free assay) 60 nM
In vitro	GSK J4 HCl is an ethyl ester derivative of the JMJD3 selective histone demethylase inhibitor GSK-J1 with an IC50 value greater than 50 μM in vitro. This compound is used to probe the consequences of demethylation of H3K27me3. In human primary macrophages, this chemical inhibits the lipopolysaccharide-induced production of cytokines, including pro-inflammatory tumour necrosis factor (TNF). In addition, it prevents the lipopolysaccharide-induced loss of H3K27me3 associated with the TNF transcription start sites and blocked the recruitment of RNA polymerase II.
Kinase Assay	Histone Demethylase AlphaScreen
Kinase Assay	Inhibition of histone demethylases is assessed using the histone demethylase AlphaScreen assay (Amplified Luminescence Proximity Homogenous Assay). This assay uses a biotinylated peptide substrate and relies on detection of the product methyl mark using a specific antibody coupled to protein-A acceptor beads and a Steptavidin donor bead to capture the peptide. In brief, recombinant demethylase enzymes are incubated in the presence of Fe2+ in the form of Ferrous Ammonium Sulphate (FAS), α-ketoglutarate (KG) and biotinylated peptide substrate. L-Ascorbic Acid is included to provide a reducing environment and prevent oxidation of Fe2+. After incubation with peptide substrate the presence of the product is detected using AlphaScreen technology. The demethylase AlphaScreen assays are performed in 384-well plate format using white proxiplates. All steps are carried out in assay buffer (50 mM HEPES pH 7.5, 0.1% (w/v) BSA and 0.01 % (v/v) Tween-20). FAS is dissolved fresh each day in 20 mM HCl to a concentration of 400 mM and diluted to 1.0 mM in deionized water. All other components are dissolved fresh each day in deionized water. For IC50 determinations 5 μL of assay buffer containing demethylase enzyme is transferred to wells of a 384-well proxiplate. Titrations of this compound (0.1 μL) are transferred to each well and the enzymes allowed to pre-incubate for 15 minutes with this chemical (final concentration of DMSO is 1%). The enzyme reaction is initiated by addition of 5 μL of a substrate mix consisting of α-KG, FAS, L-Ascorbic Acid and biotinylated peptide substrate and the reaction incubated for the indicated time at room temperature. The enzyme reaction is stopped after the indicated time by addinton of 5 μL of EDTA (7.5 mM final concentration in assay buffer). Streptavidin Donor beads (0.08 mg/ml) and Protein-A conjugated acceptor beads (0.08 mg/ml) are pre-incubated for 1 hour with an antibody to the product methyl mark and the presence of biotin-H3-product is detected by addition of 5 μL of the preincubated AlphaScreen beads (final concentrations of 0.02 mg/ml with respect to acceptor and donor beads). Detection is allowed to proceed for 1 hour at room temperature and the assay plates read in a BMG Labtech Pherastar FS plate reader. Data are normalized to the no enzyme control and the IC50 determined from the nonlinear regression curve fit using GraphPad Prism 5.
In vivo	GSK-J4 hydrochloride is a potent dual inhibitor of H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A. It inhibits LPS-induced TNF-α production in human primary macrophages. This compound is a cell permeable prodrug of GSK-J1.
References	[1] https://pubmed.ncbi.nlm.nih.gov/22842901/ [2] https://pubmed.ncbi.nlm.nih.gov/29227285/

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GSK-J4 Hydrochloride H3K27 Histone Demethylase Inhibitor

Chemical Structure

Molecular Weight: 453.96