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.

GSK-3α:10 nM (cell free), GSK-3β:6.7 nM (cell free)

方法：小鼠干细胞 ES-D3 用 CHIR-99021 (1-10 µM) 处理 72 h，使用 MTT 方法检测细胞生长抑制情况。
结果：CHIR-99021 剂量依赖性地抑制 ES-D3 细胞生长，IC50 为 4.9 µM。[1]
方法：小鼠胚胎干细胞 J1 mESCs 和小鼠胚胎瘤细胞 F9 mEC 用 CHIR-99021 (3 μM) 处理 24 h，使用 immunofluorescence 方法检测靶点蛋白表达水平。
结果：CHIR-99021 处理后，J1-mESCs 和 F9-mEC 细胞的细胞质和细胞核中的β-连环蛋白增加。[2]
方法：人 Tenon 成纤维细胞 HTFs 用 CHIR-99021 (5 μM) 处理 48 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：CHIR-99021 处理使活性形式的 GSK-3β (p-GSK-3β (Y216)) 的产生显著减少。[3]

方法：为检测体内抗肿瘤活性，将 CHIR-99021 (37.5 mg/kg/第 0-3、6-10、13-17 和 20 天每天两次) 口服给药和 paclitaxel (10 mg/kg/第 0 天单次给药) 腹腔注射给携带人非小细胞肺癌肿瘤 H1975 的 Balb/c nude 小鼠。
结果：CHIR-99021 和 paclitaxel 在体内协同作用，抑制 NSCLC 肿瘤的生长。[4]
方法：为研究 GSK-3 的直接药理学抑制是否会改变酒精在小鼠体内的积极增强作用，将 CHIR-99021 (1-10 mg/kg) 单次腹腔注射给有酒精或蔗糖自给药史的 C57BL/6J 小鼠。
结果：CHIR-99021 剂量依赖性地增加了酒精强化反应，而对蔗糖自我给药或运动活性没有影响。CHIR-99021 显著降低了 pGSK-3β 在所有测试脑区的表达，仅在 NAcb 中降低了 PICK1 并增加了 GluA2 的总表达。[5]

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].

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].

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].