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.