Figure 4.

APC2 is up-regulated by AQB, resulting in degradation of β-catenin and suppression of Wnt/β-catenin signaling. A) The ChIP products were analyzed by agarose gel electrophoresis to determine the H3K27me3-binding sites. The long upper line represents the 2,000bp upstream of the transcription initiation site. The arrow indicates the direction of transcription. The three shorter lines represent the three predicted H3K27me3-binding sites. B) C) Following treatment with DMSO or 40nM AQB for 24 hours, H3K27me3, RNA pol II phospho-Ser2 ChIP, EZH2 and H3 ChIP assays were performed. D) RT-qPCR shows mRNA levels of APC2 in the cells transfected with HOTAIR or treated with AQB (40nM) or both. E) Immunofluorescence assay displays the APC2, β-catenin and p-β-catenin levels, as well as cellular distribution after the treatment with 200nM AQB for 48 hours. DAPI was used to stain the nuclei. Scale bar, 40μm. F) Western blot analysis of β-catenin and p-β-catenin levels in the nucleus and the cytosol lysates after AQB treatment. G) Western blot analysis of Wnt/β-catenin target genes and mesenchymal markers in a panel of cancer cell lines after treatment with AQB or DMSO. H) Confocal microscopy analysis of F-actin cytoskeleton, ZEB1 and Vimentin after treatment with AQB or DMSO. Bar, 40μm. I) Transwell assay shows inhibition of cell invasion by AQB in indicated cancer cell lines. Data are presented as mean ± s.d.; n = 3 independent experiments. ***P<0.0001, **P<0.0001.