Figure 6.

FLCN knockdown causes activation of canonical Wnt signalling. (A) Knockdown of FLCN results in activation of β-catenin. Western blot on 30 µg of WCL from treated IMCD3 cells in 2D culture. Cells were harvested at 60 h post transfection. Cells were serum starved for 18 h prior to harvesting. IMCD3 express equal amounts of total β-catenin (middle panel) upon treatment with non-targeting SiCON or SiFlcn or treatment with 20 mm KCl or LiCl for 16 h. Using an antibody to detect the unphosphorylated, active β-catenin, cells treated with LiCl (an activator of β-catenin through inhibition of GSK3β) showed increased active β-catenin as did SiFlcn treated cells (upper panel). The β-tubulin (lower panel) was used as a loading control. (B) Activation of downstream targets of the Wnt pathway upon FLCN knockdown. The activation of the Wnt pathway was further confirmed using quantitative RT-PCR of downstream target genes Axin2 and CyclinD1. Upon Flcn knockdown, mRNA levels of both genes exhibited up-regulation.