Cells are treated with EPZ-6438 (1 μM) or GSK126 (1 μM) for 6 days. Protein levels are analyzed by immunoblotting. Cells are treated with EPZ-6438 (1 μM), JQ1 (0.25 μM) alone or combination for 6 days. Protein levels are analyzed by immunoblotting.
GSK126 purchased from MedChemExpress. Usage Cited in:
J Exp Med. 2018 May 7;215(5):1365-1382.
[Abstract]
Immunoblot analysis of Ezh2, H3K27me3, H3, and Hsp60 (loading control) in whole-cell lysates of the indicated proinflammatory genes of macrophages or microglia that are pretreated with DMSO or GSK126 (4 μM) for 3 d and then left nontreated (NT) or stimulated for 6 h with the ligands of different TLRs.
GSK126 purchased from MedChemExpress. Usage Cited in:
Cell Death Dis. 2018 Jan 26;9(2):129.
[Abstract]
The effects of treatment with the indicated epigenetic inhibitors on cleaved PARP (Clv-PARP) expression in both PC9/ER and HCC827/ER cells. β-actin is used as a loading control.
This includes ATF3 (Activating Transcription Factor 3) and metastasis suppressor NDRG1 (N-Myc Downstream Regulated Gene 1) that also shows an upregulation in protein expression (phosphorylated at threonine 346) with GSK126 in two EZH2 mutant cell lines by western blot.
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GSK126 (GSK2816126A) is a potent, highly selective inhibitor of EZH2 methyltransferase with an IC50 of 9.9 nM[1].
IC50 & Target[1]
EZH2
9.9 nM (IC50)
In Vitro
GSK126 potently inhibits both wild-type and mutant EZH2 methyltransferase activity with similar potencies (Ki=0.5-3 nM) independent of substrate used, and is competitive with S-adenosyl-methionine (SAM) and non-competitive with peptide substrates. GSK126 is highly selective against other methyltransferases and multiple other protein classes (EZH1, IC50=680 nM)[1]. Treatment of three SCLC cell lines with GSK126, induces growth inhibition. SCLC cell lines (Lu130, H209, and DMS53) are treated with 0.5, 2, and 8 μM GSK126, and growth curve is analyzed by WST-8 assay. Inhibition of cellular growth by GSK126 treatment is observed at 8 μM in all the three cell lines, while Lu130 and H209 are more sensitive to GSK126, even at lower doses[2].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
GSK126 Related Antibodies
In Vivo
GSK126 is administered intraperitoneally at a dose volume of 0.2 mL per 20 g body weight in female beige SCID mice. GSK126 effectively inhibits the proliferation of EZH2 mutant DLBCL cell lines and markedly inhibits the growth of EZH2 mutant DLBCL xenografts in mice[1].
MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.
Room temperature in continental US; may vary elsewhere.
Storage
Powder
-20°C
3 years
4°C
2 years
In solvent
-80°C
2 years
-20°C
1 year
Solvent & Solubility
In Vitro:
DMSO : 12.5 mg/mL (23.73 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)
Preparing Stock Solutions
ConcentrationSolventMass
1 mg
5 mg
10 mg
1 mM
1.8987 mL
9.4936 mL
18.9872 mL
5 mM
0.3797 mL
1.8987 mL
3.7974 mL
10 mM
0.1899 mL
0.9494 mL
1.8987 mL
View the Complete Stock Solution Preparation Table
*Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles. Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.
For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day. The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.
Protocol 1
Add each solvent one by one: 10% DMSO 40% PEG300 5% Tween-80 45% Saline
This protocol yields a clear solution of ≥ 1.25 mg/mL (saturation unknown).
Taking 1 mL working solution as an example, add 100 μLDMSO stock solution (12.5 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.
Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
Protocol 2
Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: 1.25 mg/mL (2.37 mM); Suspended solution; Need ultrasonic
This protocol yields a suspended solution of 1.25 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.
Taking 1 mL working solution as an example, add 100 μLDMSO stock solution (12.5 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
Protocol 3
Add each solvent one by one: 10% DMSO 90% Corn Oil
This protocol yields a clear solution of ≥ 1.25 mg/mL (saturation unknown). If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Taking 1 mL working solution as an example, add 100 μLDMSO stock solution (12.5 mg/mL) to 900 μLCorn oil, and mix evenly.
Protocol 4
Add each solvent one by one: 5% DMSO 40% PEG300 5% Tween-80 50% Saline
For the following dissolution methods, please prepare the working solution directly.
It is recommended to prepare fresh solutions and use them promptly within a short period of time. The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution.
If precipitation or phase separation occurs during preparation,
heat and/or sonication can be used to aid dissolution.
Protocol 1
Add each solvent one by one: 50% PEG300 50% Saline
Solubility: 10 mg/mL (18.99 mM); Suspended solution; Need ultrasonic
Protocol 2
Add each solvent one by one: 20% SBE-β-CD in Saline adjusted to pH 4-4.5 with 1 N Acetic
Solubility: 20 mg/mL (37.97 mM); Clear solution; Need ultrasonic and warming
In Vivo Dissolution Calculator
Please enter the basic information of animal experiments:
Dosage
mg/kg
Animal weight (per animal)
g
Dosing volume (per animal)
μL
Number of animals
Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
Please enter your animal formula composition:
%
DMSO+
%
+
%
Tween-80
+
%
Saline
Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
The co-solvents required include: DMSO,
. All of co-solvents are available by MedChemExpress (MCE).
, Tween 80. All of co-solvents are available by MedChemExpress (MCE).
Calculation results:
Working solution concentration:
mg/mL
Method for preparing stock solution:
mg
drug dissolved in
μL
DMSO (Stock solution concentration: mg/mL).
The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
Method for preparing in vivo working solution for animal experiments: Take
μL DMSO stock solution, add
μL .
μL , mix evenly, next add
μL Tween 80, mix evenly, then add
μL Saline.
Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution
If the continuous dosing period exceeds half a month, please choose this protocol carefully.
Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
[1]. McCabe MT, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012 Dec 6;492(7427):108-12.
[Content Brief]
[2]. Sato T, et al. PRC2 overexpression and PRC2-target gene repression relating to poorer prognosis in small cell lung cancer. Sci Rep. 2013 May 29;3:1911.
[Content Brief]
Kinase Assay
[1]
The five-member PRC2 complex (Flag-EZH2, EED, SUZ12, AEBP2, RbAp48) containing either wild-type or mutant (A677G, Y641N, Y641C, Y641H, Y641S or Y641F) EZH2 is prepared. GSK126 is dissolved in DMSO and tested at concentrations of 0.6 nM to 300 nM with a final DMSO concentration of 2.5%. In contrast to wild-type EZH2 which prefers H3K27me0 as a substrate in vitro, EZH2 Y641 mutants prefer H3K27me2 and have little activity with H3K27me0 or H3K27me1. The A677G mutant is distinct from both the wild-type and Y641 mutant forms of EZH2 in that it efficiently methylates H3K27me0, H3K27me1, and H3K27me2; therefore, histone H3 peptides (residues 21-44; 10 μM final) with either K27me0 (wild type, A677G EZH2), K27me1 (A677G EZH2), or K27me2 (A677G, Y641N, Y641C, Y641H, Y641S and Y641F EZH2) are used as methyltransferase substrates. GSK126 is added to plates followed by addition of 6 nM EZH2 complex and peptide. As the potency of GSK126 is at or near the tight binding limit of an assay run at [SAM]=Km, IC50 values are measured at a high concentration of the competitive substrate SAM relative to its Km (7.5 μM SAM where the SAM Km is 0.3 μM)[1].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Cell Assay
[2]
JUB- and PTRF-introduced DMS53 cells are seeded at density of 1×103 cells/well in 96-well plate, and cellular growth is analyzed using WST-8 kit at 12, 36, 60, and 84 h. Cellular growth of Lu130, H209, and DMS53 with treatment by DZNep or GSK126 is also analyzed using WST-8 kit. DZNep is dissolved in PBS at 5 mM, and cells are cultured at the final concentration of 5 μM. GSK126 is dissolved in DMSO at 10 mM, and cells are cultured at 0.5, 2, and 8 μM[2].
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
Animal Administration
[1]
Mice[1] GSK126 or vehicle is administered intraperitoneally at a dose volume of 0.2 mL per 20 g body weight. Pfeiffer or KARPAS-422 cells (1×107) in 100% Matrigel are implanted subcutaneously in female beige SCID mice. Tumors are measured with calipers, and block randomized according to tumour size into treatment groups. For efficacy studies, 10 mice are randomized in each treatment group before the initiation of dosing and GSK126 treatment is initiated once the tumour volumes are approximately 200 mm3 in the Pfeiffer and KARPAS-422 studies and 500 mm3 in the KARPAS-422 intermittent dosing study. Mice are weighed and tumors measured with calipers twice weekly. Two-tailed t-tests are conducted assuming two samples of equal variance.
MCE has not independently confirmed the accuracy of these methods. They are for reference only.
References
[1]. McCabe MT, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012 Dec 6;492(7427):108-12.
[Content Brief]
[2]. Sato T, et al. PRC2 overexpression and PRC2-target gene repression relating to poorer prognosis in small cell lung cancer. Sci Rep. 2013 May 29;3:1911.
[Content Brief]
[1]. McCabe MT, et al. EZH2 inhibition as a therapeutic strategy for lymphoma with EZH2-activating mutations. Nature. 2012 Dec 6;492(7427):108-12.
[2]. Sato T, et al. PRC2 overexpression and PRC2-target gene repression relating to poorer prognosis in small cell lung cancer. Sci Rep. 2013 May 29;3:1911.
Complete Stock Solution Preparation Table
*Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles. Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.
Optional Solvent
ConcentrationSolventMass
1 mg
5 mg
10 mg
25 mg
DMSO
1 mM
1.8987 mL
9.4936 mL
18.9872 mL
47.4681 mL
5 mM
0.3797 mL
1.8987 mL
3.7974 mL
9.4936 mL
10 mM
0.1899 mL
0.9494 mL
1.8987 mL
4.7468 mL
15 mM
0.1266 mL
0.6329 mL
1.2658 mL
3.1645 mL
20 mM
0.0949 mL
0.4747 mL
0.9494 mL
2.3734 mL
GSK126 Related Classifications
Epigenetics
Histone Methyltransferase
Help & FAQs
Do most proteins show cross-species activity?
Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.
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