JIB-04

For research use only. Not for use in humans.

製品コードS7281 別名:NSC 693627

JIB-04化学構造

分子量(MW):308.76

JIB-04 is a pan-selective Jumonji histone demethylase inhibitor with IC50 of 230, 340, 855, 445, 435, 1100, and 290 nM for JARID1A, JMJD2E, JMJD3, JMJD2A, JMJD2B, JMJD2C, and JMJD2D in cell-free assays, respectively.

サイズ 価格(税別) 在庫  
JPY 34700 あり
JPY 63400 あり
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バルク問合せ

カスタマーフィードバック(2)

  • G, Western blot analyses to monitor the amount of H3K9me3 in the presence of rhein and MMS (upper panel) and under the treatment of JIB-04 (lower panel).

    The journal of biological chemistry, 2016, 291(21):11083-11093.. JIB-04 purchased from Selleck.

    Immunofluorescence staining determined XRCC1and γH2AX foci by exposure of SGC7901/DDP cells to 5μg/ml cisplatin and 10μM JIB-04 for 24h (×1000).

    Int J Biol Sci, 2018, 14(9):1122-1132. JIB-04 purchased from Selleck.

製品安全説明書

Histone Demethylase阻害剤の選択性比較

生物活性

製品説明 JIB-04 is a pan-selective Jumonji histone demethylase inhibitor with IC50 of 230, 340, 855, 445, 435, 1100, and 290 nM for JARID1A, JMJD2E, JMJD3, JMJD2A, JMJD2B, JMJD2C, and JMJD2D in cell-free assays, respectively.
ターゲット
JARID1A [1]
(Cell-free assay)
JMJD2D [1]
(Cell-free assay)
JMJD2E [1]
(Cell-free assay)
JMJD2B [1]
(Cell-free assay)
JMJD2A [1]
(Cell-free assay)
230 nM 290 nM 340 nM 435 nM 445 nM
体外試験

JIB-04 induces transcriptional changes in a cancer-selective manner, including the downregulation of proliferative genes and the upregulation of the anti-proliferative/pro-apoptotic genes. JIB-04 blocks growth of lung and prostate cancer lines with IC50 as low as 10 nM, while produces less anti-proliferative activities on HBECs and PrSCs/PrECs. [1]

アッセイ
Methods Test Index PMID
Western blot
H3K9me2 / H3K9me3 / H3K27me3 / H3K4me3 / TIMP1 / ANP ; 

PubMed: 30531796     


Western blots of indicated proteins from lysates of NRVMs (neonatal rat ventricular myocytes) (b) and human iPSC-CM (c) treated with various concentration of JIB-04.

αCD133; 

PubMed: 29700375     


Protein expression of CD133 in colorectal cancer cells after treatment with 2 µM JIB-04 for 24 h was confirmed by western blot analysis. GAPDH was used as a loading control. 

30531796 29700375
Immunofluorescence
DNA-PKcs; 

PubMed: 30355483     


DNA-PKcs p-T2609 foci kinetics in H1299 cells. Cells were incubated with vehicle or 16 nM JIB-04 for 4 hr, irradiated (10 Gy), fixed, and immunostained, and then the number of foci per nucleus in >100 cells was counted for each time point.

30355483
Growth inhibition assay
Cell viability; 

PubMed: 30237855     


One day following plating, the indicated cells (7 different Ewing Sarcoma cell lines, and human mesenchymal stem cells (hMSC)) were treated for 48 hours with the indicated concentrations of JIB-04. Cell numbers at the end of the experiment were quantified using an MTT assay, and were normalized to vehicle-treated cells. Results represent the mean and standard error of the mean (SEM) of at least 2 independent experiments, each performed in replicate. IC50 values for growth/survival inhibition of Ewing Sarcoma cells by JIB-04, calculated from the data in panel A, are shown on right.

30237855
体内試験 In two separate xenograft mouse models (H358 or A549), JIB-04 diminishes tumor growth, lowers Jumonji histone demethylase activity in tumors, and prolongs cancer survival. [1]

お薦めの試験操作(参考用のみ)

キナーゼ試験:

[1]

- 合併

Jumonji demethylase/prolyl hydroxylase/LSD1 activity assays:

Active JMJD2E aa 1-350 is purified from E.coli and used in vitro in the presence of α-ketoglutarate, 5-10 μM iron, ascorbic acid and a histone peptide substrate in a coupled reaction with formaldehyde dehydrogenase supplemented with NAD+ to quantify NADH production or using Epigentek kit P-3081. For histone demethylation reactions quantified by Western analysis, a His-tagged hJMJ2D aa 1-350 expression construct, the kind gift of Drs. Y. Shi and J. Whetstine, is expressed and purified from E.coli following the Qiagen Ni-NTA agarose manual instructions and the protocol of Whestine et al 54. Briefly, protein is eluted in 50 mM TrisHCl pH 7.8 + 0.3 M NaCl + 10% Glycerol + 200 mM immidazol, and dialyzed against 20 mM TrisHCl pH 7.4, 0.15 M NaCl, 0.2 mM PMSF, 0.5 mM DTT, 8% glycerol. Enzyme is aliquoted, flash frozen and stored at -80°C. For activity assays by Western blot, ~1.5 μg of enzyme are combined with 0.3 μg H3K9me3 substrate (Active Motif #31213) in 10 μM (NH4)2Fe(SO4)2, 1 mM α-ketoglutarate, and 2 mM sodium L-ascorbate in 50 mM Hepes pH 7.9 in the presence of vehicle or drug and incubated for 30 min-2 hrs at 37°C. SDS loading buffer is added to the reactions, and after boiling, samples are run on NuPage 4-12% Bis-Tris gels, transferred to nitrocellulose and blotted using Upstate #07-523 to detect H3K9me3. For the detection of H3 total signal, we uses Active Motif #39763 (primary) and IRDye 680 conjugated donkey anti-rabbit IgG (secondary, LI-COR # 926-32223) and imaged blots in an Odyssey Infrared Imaging system kindly made available by Dr. M. Cobb. For in vitro IC50 determinations and competition studies, typically 100-200 ng of purified protein are incubated with vehicle, JIB-04 or analogs, as indicated in figure legends and activity measured by ELISA (Epigentek kit P-3081 for H3K9me3 demethylation, P-3083 for H3K4me3 demethylation, and P-3085 for H3K27me3 demethylation) in reactions containing 50mM Hepes pH 7.5, 0.01% Tween 20, 120nM (NH4)2Fe(SO4)2, 1 mM α-ketoglutarate, 2 mM sodium L-ascorbate and 50ng peptide substrate. Final enzyme concentrations in the reactions were as follows: 206 nM JMJD2A, 12 nM JMJD2B, 60 nM JMJD2C, 90 nM JMJD2D E.coli, 30 nM JMJD2D Sf9, 30 nM JMJD2E, 30 nM Jarid1a, 35 nM JMJD3. Background readings are given by heat inactivated enzymes, 0.5-1 mM 2,4 PDCA or reactions with no 2-OG. hJMJD2A (aa1-350) purified in E.coli is the kind gift of Dr. Jose Rizo-Rey and is assayed at 400 ng/reaction due to its intrinsic low activity. GraphPad Prism software is used for IC50 calculations and curve fitting. E.coli JMJD2D purified by us and Sf9 JMJD2D from BPS gave undistinguishable results. Note that for substrate competition assays, in order to remain in the linear range of the assay and not saturate binding capacity of the ELISA plate, reactions with > 0.75μM H3K9me3 containes unbiotinylated substrate or are diluted at the detection step and signals adjusted per dilution factor. For the direct quantification of H3K9me3 demethylase activity in cell lysates, treated cells (plated at 2 million/10cm dish) or tumor homogenates in PBS were sonicated (3x 4 sec) and equal amounts of protein are incubated with a histone H3K9me3 substrate in a reaction buffer containing cofactors for 2h at 37°C before specific immune-detection of the H3K9me2 product using Epigentek kit P-3081 reagents. 500 ng of E.coli purified PHD2 protein in 40mM Tris pH 7.4, 100mM NaCl, 20% glycerol, 5mM β-mercapto-ethanol, 10mM maltose are used to obtain activity in the linear range. Biotinylated peptides derived from the HIF-1 ODD (Biotin-Acp-DLDLEALAPYIPADDDFQL or Biotin-Acp-DLDLEALAP(OH)YIPADDDFQL as a hydroxylated control) are immobilized on Neutravidin-coated 96-well plates. Enzyme is incubated in the coated wells in reaction buffer (20 mM Tris-Cl pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 2 mM DTT, 0.12 μM ferrous sulfate, 0.5 mM 2-oxoglutarate and 1 mM ascorbate) for 45 min at room temperature in the presence of the indicated drugs. The competitive analog of α-ketoglutarate, DMOG, is used as a positive control for inhibition. Peptide hydroxylation is detected using a polyclonal rabbit antibody raised against a hydroxylated HIF peptide epitope, (rabbit anti-hydroxyproline 4817, made in house), followed by addition of a goat anti-rabbit HRP-conjugated secondary antibody. Luminescence is measured in an EnVision plate reader. The activity of LSD1 recombinant protein is measured using Epigentek kit P-3075 according to the manufacturer’s protocol with the proprietary inhibitor.
細胞試験:

[1]

- 合併
  • 細胞株: Human lung cancer cell lines (LCa), primary or immortalized non-tumorigenic HBECs, prostate cancer (PCa), primary prostate stromal (PrSC) and prostate epithelial cells (PrEC).
  • 濃度: ~10 μM
  • 反応時間: 96 hours
  • 実験の流れ:

    For cell viability assays, cells are plated at 1500-3000 cells/well in 96 well plates and treated the next day with increasing doses of compound over 4 days and their viability assessed by standard MTS assays using Promega’s Cell Titer or Cell Titer-Glo reagents according to the manufacturer’s protocols. Absorbance at 490 nm and 650 nm or luminescence is measured by a Spectra Max or a FlouroStar Omega plate reader. Data are normalized to the untreated controls (100% viability). Each cell line is tested in 2-5 independent assays, each containing 4-8 replicates. IC50 values are calculated using DIVISA, a high-throughput software, developed in hous, for storing and analyzing drug sensitivity assays. Dose-response curves are plotted using a non-linear regression model and IC50s are determined from the fitted curves. The average IC50 derived from 2-5 independent assays, each containing 4-8 replicates is reported.


    (参考用のみ)
動物試験:

[1]

- 合併
  • 動物モデル: Mice harboring H358 xenografts or A549 xenografts
  • 投薬量: 110 mg/kg (H358, i.p.), 55 mg/kg (A549, Oral gavage)
  • 投与方法: Oral gavage or i.p.
    (参考用のみ)

溶解度 (25°C)

体外 DMSO 25 mg/mL (80.96 mM) warming
Water Insoluble
Ethanol Insoluble
体内 左から(NMPから)右の順に溶剤を製品に加えます(文献ではなく、Selleckの実験によるデータ):
4% DMSO+corn oil
混合させたのち直ちに使用することを推奨します。
3mg/mL

* 溶解度測定はSelleck技術部門によって行われており、その他文献に示されている溶解度と差異がある可能性がありますが、同一ロットの生産工程で起きる正常な現象ですからご安心ください。

化学情報

分子量 308.76
化学式

C17H13ClN4

CAS No. 199596-05-9
保管
in solvent
別名 NSC 693627
Smiles ClC1=CN=C(N/N=C(C2=CC=CC=C2)/C3=NC=CC=C3)C=C1

投与溶媒組成計算器(クリア溶液)

ステップ1:実験データを入力してください。(余分な消耗を考慮し動物一匹分の量を用意することをお勧めします。)
投与量 mg/kg 動物平均体重 g 投与体積(動物毎) ul 動物数
ステップ2:投与溶媒の組成を入力してください。(ロットごとに組成が異なるため、セレックから完全に溶解できる組成をお求めください。)
% DMSO % % Tween 80 % ddH2O
計算リセット

便利ツール

モル濃度計算器

モル濃度計算器

求めたい質量、体積または濃度を計算してください。

質量 (mg) = 濃度 (mM) x 体積 (mL) x 分子量 (g/mol)

モル濃度計算器方程式

  • 質量
    濃度
    体積
    分子量

*貯蔵液を準備するとき、常に、オンであるとわかる製品のバッチに特有の分子量を使って、を通してラベルとMSDS/COA(製品ページで利用可能な)。

希釈計算器

希釈計算器

貯蔵液を準備するために必要な希釈率を計算してください。Selleck希釈計算器は、以下の方程式に基づきます:

開始濃度 x 開始体積 = 最終濃度 x 最終体積

希釈の計算式

この方程式は、一般に略語を使われます:C1V1 = C2V2 ( 入力 出力 )

  • C1
    V1
    C2
    V2

常に貯蔵液を準備するとき、小びんラベルとMSDS/COA(オンラインで利用できる)で見つかる製品のバッチに特有の分子量を使ってください。

連続希釈計算器方程式

  • 連続希釈剤

  • 計算結果

  • C1=C0/X C1: LOG(C1):
    C2=C1/X C2: LOG(C2):
    C3=C2/X C3: LOG(C3):
    C4=C3/X C4: LOG(C4):
    C5=C4/X C5: LOG(C5):
    C6=C5/X C6: LOG(C6):
    C7=C6/X C7: LOG(C7):
    C8=C7/X C8: LOG(C8):
分子量計算器

分子量计算器

そのモル質量と元素組成を計算するために、合成物の化学式を入力してください:

総分子量:g/mol

チップス: 化学式は大文字と小文字の区別ができます。C10H16N2O2 c10h16n2o2

モル濃度計算器

質量 濃度 体積 分子量

技術サポート

ストックの作り方、阻害剤の保管方法、細胞実験や動物実験の際に注意すべき点など、製品を取扱う時に問い合わせが多かった質問に対しては取扱説明書でお答えしています。

Handling Instructions

他に質問がある場合は、お気軽にお問い合わせください。

  • * 必須

よくある質問(FAQ)

  • 質問1:

    Do you have any suggestion for in vivo study please? (Cat. S7281, via injection)

  • 回答:

    For IP or sub cutaneous injection, The compound is soluble in 4% DMSO+30% PEG 300+5% Tween 80+ddH2O at 3 mg/ml. But when water added, it turned yellowish green immediately. We are not sure if the compound was still fine in this vehicle. So we tried to use oil. 3mg of this compound dissolves in 40ul of DMSO clearly, and it can be dilute with corn oil at any proportion.

Histone Demethylaseシグナル伝達経路

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細胞株 試験類型 濃度 培養時間 溶剤類型 活性叙述 PMID