IM-12, an effective GSK-3β inhibitor(IC50=53 nM), regulates Wnt signalling.

GSK-3β:53 nM

在hNPCs中（IC50=3.8 μM）,IM-12能够抑制GSK-3β活性,增加β-连环蛋白浓度。在人神经祖细胞中,IM-12通过对于Wnt信号通路的激活,促进细胞增殖。

GSK-3β activity assay: IC50 of new synthesised compound IM-12 to GSK-3β is determined by a luminometric GSK-3β activity assay. Briefly, compounds are tested in different concentrations diluted in assay buffer containing final concentrations of: 4 mM MOPS pH 7.2; 0.4 mM EDTA; 1 mM EGTA; 2.5 mM β-glycerophosphate; 4 mM MgCl2; 40 μM BSA; 0.05 mM DTT. Four microlitres of diluted compounds are added to 25 μM pGS-2 peptide substrate, 20 ng recombinant GSK-3β and 1 μM ATP to a total assay volume of 40 μl. The enzymatic reaction is stopped after 30 min of incubation at 30 °C by adding 40 μl KinaseGlo. The luminometric signal is allowed to stabilise for 10 min and the measured with a Glomax? 96 Microplate Luminometer.

To measure viable cells, 50-100 μL of cell suspension is analyzed using CASY technology with the appropriate program. ReNcell VM cells are seeded at a defined cell number and proliferated for 24 h. Then the medium is changed to proliferation medium with added substances at indicated concentrations. The cell number was determined every 24 h. Cells were exposed to the added drugs during the whole experiment, whereas the media is changed every 24 h.(Only for Reference)