Figure 3. ERG increases activeβ-cateninin prostate cancer.

A. Active β-catenin is increased by ERG overexpression. HEK293T cells were transfected with ERG or β-catenin plasmid. The levels of total and active β-catenin were determined by western blot analysis.

B. ERG knockdown decreased active β-catenin level. VCaP cells were subjected to ERG knockdown using shRNAlentivirus to generate stable cells. Cells were analyzed by western blotting using active β-catenin antibody.

C. ERG overexpression induces AKT and GSK-3β phosphorylation. Prostate cell lines with stable expression of control or ERG gene were analyzed by immunoblotting.

D–E. ERG and β-catenin synergistically induce Wnt signaling. HEK293T (D) or 22Rv1 (E) cells were transfected with ERG and/or β-catenin, along with SuperTOPFlash luciferase reporter construct and the control Renilla expression plasmid. Luciferase activities was assayed at 48 h post-transfection and normalized against Renilla internal control values. Error bars indicate triplicate experiments, mean ± SEM.

F. AXIN2 is up-regulated by ERG. Prostate cell lines including BPH1, RWPE and 22Rv1 were infected with ERG-expressing construct and selected for stable overexpression. QRT-PCR was carried out to assay transcript levels.

G. AXIN2 is down-regulated following ERG knockdown. Stable ERG knockdown was generated in VCaP cells using shRNAlentivirus followed by colony selection.