| 名稱 | CHIR-99021 |
| 描述 | CHIR-99021 (CT99021) is an activator of the Wnt/β-catenin signaling pathway and a GSK-3α/β inhibitor (IC50=10/6.7 nM) with selective and oral activity.CHIR-99021 induces cellular autophagy, which enhances self-renewal in mouse and human embryonic stem cells. |
| 細胞實驗 | The Wnt/beta-catenin reporter assay was performed with the M50 Super 8× TOPFlash and M51 Super 8× FOPFlash vector containing the firefly luciferase gene under the control of TCF/LEF binding sites or mutated bindings sites. 12,500 cells were seeded overnight on gelatine-coated 96-well plates in LIF-containing ES cell medium. On the next day, the cells were transfected using Lipofectamine with one of the aforementioned vectors plus pGL4.75 [hRluc/CMV] encoding the renilla luciferase reporter gene hRluc as a transfection control. Six hours after transfection the medium was changed to medium devoid of LIF, with reduced serum, and supplemented with 5 μM CHIR-99021. The Dual-Luciferase? reporter assay system was employed 48 and 72 h after the medium change to follow the luminescence reaction using a GloMax?-multi detection system [4]. |
| 激酶實驗 | Kinases were purified from SF9 cells through the use of their His or Glu tag. Glu-tagged proteins were purified as described, and His-tagged proteins were purified according to the manufacturer's instructions. Kinase assays were performed in 96-well plates with appropriate peptide substrates in a 300-μl reaction buffer (variations on 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 25 mMβ-glycerophosphate, 1 mM NaF, and 0.01% bovine serum albumin). Peptides had Km values from 1 to 100 μM. CHIR 99021 or CHIR GSKIA was added in 3.5 μl of Me2SO, followed by ATP to a final concentration of 1 μM. After incubation, triplicate 100-μl aliquots were transferred to Combiplate 8 plates containing 100 μl/well of 50 μM ATP and 20 mM EDTA. After 1 hour, the wells were rinsed five times with phosphate-buffered saline, filled with 200 μl of scintillation fluid, sealed, and counted in a scintillation counter 30 min later. All of the steps were at room temperature. The percentage of inhibition was calculated as 100 × (inhibitor ? no enzyme control)/(Me2SO control ? no enzyme control) [4]. |
| 動物實驗 | Blood was obtained by shallow tail snipping at lidocaine-anesthetized tips. Blood glucose was measured directly or heparinized plasma was collected for measurement of glucose or insulin. Animals were pre-bled and randomized to vehicle control or GSK-3 inhibitor treatment groups. For glucose tolerance tests (GTTs), animals fasted throughout the procedure with food removal early in the morning, 3 h before the first prebleed (db/db mice), or the previous night, 16 h before the bleed (ZDF rats). When the time course of plasma glucose and insulin changes in fasting ZDF rats was measured, food was removed ～16 h before test agent administration. The glucose challenges in the GTT were 1.35 g/kg i.p. (ipGTT) or 2 g/kg via oral gavage (oGTT). CHIR-99021 were formulated as solutions in 20 mmol/l citrate-buffered 15% Captisol or as fine suspensions in 0.5% carboxymethylcellulose [1]. |
| 體外活性 | 方法：小鼠干細胞 ES-D3 用 CHIR-99021 (1-10 μM) 處理 72 h，使用 MTT 方法檢測細胞生長抑制情況。
結(jié)果：CHIR-99021 劑量依賴性地抑制 ES-D3 細胞生長，IC50 為 4.9 μM。[1]
方法：小鼠胚胎干細胞 J1 mESCs 和小鼠胚胎瘤細胞 F9 mEC 用 CHIR-99021 (3?μM) 處理 24 h，使用 immunofluorescence 方法檢測靶點蛋白表達水平。
結(jié)果：CHIR-99021 處理后，J1-mESCs 和 F9-mEC 細胞的細胞質(zhì)和細胞核中的β-連環(huán)蛋白增加。[2]
方法：人 Tenon 成纖維細胞 HTFs 用 CHIR-99021 (5 μM) 處理 48 h，使用 Western Blot 方法檢測靶點蛋白表達水平。
結(jié)果：CHIR-99021 處理使活性形式的 GSK-3β (p-GSK-3β (Y216)) 的產(chǎn)生顯著減少。[3] |
| 體內(nèi)活性 | 方法：為檢測體內(nèi)抗腫瘤活性，將 CHIR-99021 (37.5 mg/kg/第 0-3、6-10、13-17 和 20 天每天兩次) 口服給藥和 paclitaxel (10 mg/kg/第 0 天單次給藥) 腹腔注射給攜帶人非小細胞肺癌腫瘤 H1975 的 Balb/c nude 小鼠。
結(jié)果：CHIR-99021 和 paclitaxel 在體內(nèi)協(xié)同作用，抑制 NSCLC 腫瘤的生長。[4]
方法：為研究 GSK-3 的直接藥理學抑制是否會改變酒精在小鼠體內(nèi)的積極增強作用，將 CHIR-99021 (1-10 mg/kg) 單次腹腔注射給有酒精或蔗糖自給藥史的 C57BL/6J 小鼠。
結(jié)果：CHIR-99021 劑量依賴性地增加了酒精強化反應，而對蔗糖自我給藥或運動活性沒有影響。CHIR-99021 顯著降低了 pGSK-3β 在所有測試腦區(qū)的表達，僅在 NAcb 中降低了 PICK1 并增加了 GluA2 的總表達。[5] |
| 存儲條件 | store at low temperature | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | 10% DMSO+40% PEG300+5% Tween 80+45% Saline : 0.93 mg/mL (2 mM), Please add co-solvents sequentially, clarifying the solution as much as possible before adding the next one. Dissolve by heating and/or sonication if necessary. Working solution is recommended to be prepared and used immediately.
DMSO : 50 mg/mL (107.45 mM)
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| 關鍵字 | GSK-3 | Glycogen synthase kinase 3 | CHIR99021 | β-catenin | inhibit | Autophagy | Beta catenin | CT-99021 | CHIR 99021 | Wnt | Inhibitor | Glycogen synthase kinase-3 | CT 99021 |
| 相關產(chǎn)品 | Guanidine hydrochloride | Naringin | Valproic Acid | Taurine | Gefitinib | Aceglutamide | Hydroxychloroquine | Curcumin | Stavudine | Salicylic acid | Paeonol | Sodium 4-phenylbutyrate |
| 相關庫 | 神經(jīng)保護化合物庫 | 抑制劑庫 | 經(jīng)典已知活性庫 | 抗癌化合物庫 | 已知活性化合物庫 | 抗胰腺癌化合物庫 | 激酶抑制劑庫 | 高選擇性抑制劑庫 | 抗衰老化合物庫 | NO PAINS 化合物庫 | 免疫/炎癥分子化合物庫 | 神經(jīng)退行性疾病化合物庫 |