3.2 ISX9 induces the accumulation of β-catenin without affecting the expression and activity of LRP6 and GSK3β
To explore the potential target of ISX9 action in the Wnt signaling cascade, we examine the effect of ISX9 on key components in Wnt signaling cascade, including active β-catenin, total β-catenin, phosphorylated LRP6, total LRP6, Ser9 phosphorylated GSK3β, total GSK3β, and CK1ɛ in HEK293T, HaCat and NIH3T3 cells. As shown in Figure 2, ISX9 treatment dramatically upregulated protein levels of active β-catenin and total β-catenin in a concentration-dependent fashion, whereas it had little effect on the levels of phosphorylated LRP6, total LRP6, Ser9 phosphorylated GSK3β, total GSK3β, and CK1ɛ in all three cell lines. We also checked ISX9 effect on mRNA expression of β-catenin in HEK293T cells. There was no significant change in mRNA expression of β-catenin in response to ISX9 treatment (Supplementary Figure S1). Collectively, these results imply that ISX9 could activate Wnt/β-catenin signaling mainly through inducing the accumulation of intracellular β-catenin without affecting the expression and activity of LRP6 and GSK3β.