| Description |
BIX-01294 is an inhibitor of G9a Histone Methyltransferase with IC50 of 1.9 μM.
|
| Related Catalog |
|
| Target |
IC50: 1.9 μM (G9a Histone Methyltransferase)
|
| In Vitro |
BIX-01294 (4.1 μM) treatment of G9anull ES cells results in a further reduction in H3K9me2 levels. BIX-01294 reduces H3K9me2 at several G9a target genes[1]. BIX-01294 (5 µM) reduces levels of global H3K9 methylation in pronuclear- and 2-cell-stage embryos. Embryos transiently exposed to BIX-01294 have a reduced ability to establish pregnancy[2]. BIX-01294 (1 µg/mL) causes reduction in the BrdU incorporation of fetal PASMCs. BIX-01294 treatment decreases the PASMCs migration induced by PDGF[3].
|
| Kinase Assay |
Test compounds are diluted to 12 μg/mL in 50 mM Tris-HCl pH 8.5 containing 4% DMSO and 10 μL is dispensed into the wells. Blank and control wells receive only compound buffer. GST-G9a at 10 μg/mL and SAM at 40 μM are diluted in 50 mM Tris HCl pH 8.5/10 mM DTT and added in a volume of 20 μL. Blank wells receive Tris/DTT buffer only. The reactions are initiated by the addition of 800 nM H3(1-20)-cysbiotin substrate in 50 mM Tris pH 8.5 in a volume of 10 μL, and incubated at room temperature for 60 minutes. The plates are washed 3 times with 100 μL of ish Buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween 20, 0.2% BSA). Next, 50 μL of luoroimmunoassay (FI) Buffer (50 mM Tris HCl pH 7.8, 150 mM NaCl, 0.05% Tween 40, 25 μM DTPA, 0.2% BSA, 0.05% BGG) containing 5 ng α-2X-di-meth H3-K9 and 5 ng goat anti-rabbit Eu chelate is added to all wells of the plate, and the platei s incubated for an additional hour at room temperature. The plates are washed 3 times with 100 μL of ish Buffer, and 50 μL of Enhancement Solution is added to each well. Time resolved fluorescence is measured after 45 minutes on a Viewlux Microplate Imager imaging for 15 seconds with a 354 μs window, 400 μs delay, excitation at 360 nm, and emission at 618 nm.
|
| Cell Assay |
Briefly, fetal PASMCs are plated in 96-well plates and starved for 24 h at 0.1% serum containing medium. PDGF-BB is added for 24 h at the indicated concentrations in the presence or absence of BIX-01294. BrdU label solution is added to each well 18 h prior to analysis. Denaturing solution is added to each well for 30 min at room temperature after removing the contents of wells. Then, anti-BrdU antibody is added to each well and incubated for 1 h and peroxidase goat anti-mouse IgG HRP conjugate is added in the well for 30 min at room temperature. The absorbance is read at 450-540 nm on a Glomax Multiple Detection System.
|
| References |
[1]. Kubicek S, et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol Cell. 2007 Feb 9;25(3):473-81. [2]. Park KE, et al. IVMBIX-01294, an inhibitor of the histone methyltransferase EHMT2, disrupts histone H3 lysine 9 (H3K9) dimethylation in the cleavage-stage porcine embryo. Reprod Fertil Dev. 2012;24(6):813-21. [3]. Yang Q, et al. BIX-01294 treatment blocks cell proliferation, migration and contractility in ovine foetal pulmonary arterial smooth muscle cells. Cell Prolif. 2012 Aug;45(4):335-44.
|
