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CHIR-99021 is a highly specific glycogen synthase kinase-3 (GSK-3) inhibitor which can inhibit both isoforms with IC50 of 10 nM (GSK-3α) and 6.7 nM (GSK-3β).

CHIR-99021 was proved to promote self-renewal and maintain pluripotency of both B6 and BALB/c ES cells via stabilizing the downstream effectors like c-Myc and β -catenin[1]. In J1 mESC cells, CHIR-99021 played an important role in maintaining the colony morphology as well as the self-renewal when combined with leukemia inhibitory factor (LIF). CHIR-99021 has shown to regulate multiple signaling pathways which involve Wnt/β-catenin, TGF-β, Nodal and MAPK, and the expression of epigenetic regulatory genes like Dnmt3l[2].

CHIR-99021 has been demonstrated to promote DN3 thymocytes proliferation and differentiation in the absence of pre-TCR signaling, Notch1 signaling or CXCL12[3]. However, study has also found that higher concentration (10 μM but not 1 μM or 3 μM) of CHIR99021 might selectively inhibit differentiation by activating IL-7 signaling pathway[3].

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