β-catenin knockdown reveals novel cell-cycle regulators as targets in MM. (A) Decreased Wnt reporter activity in β-catenin shRNA cells. MM1.S and OPM1 cells were transfected with TOPFLASH and assayed for luciferase activity after 48 hours. Control FOPFLASH levels were unaffected. Experiments were performed in triplicate and repeated twice. The results denote the average and SEM of triplicate assays. (B) GEP revealed 2 distinct sets of genes that are most up- or down-regulated by β-catenin knockdown compared with control shRNA MM1.S cells. (C) Microarray target validation by immunoblot show decreased expression of known and novel targets after β-catenin knockdown in MM1.S (left) and OPM1 cells (right). (D) GEP of β-catenin shRNA down-regulated genes containing LEF1/TCF4 binding sites (GCTTTGT/A). Only probe sets expressed in all 3 samples are shown. (E) Immunofluorescence of control or β-catenin shRNA MM1.S cells (100×) for AurKA (red) and DAPI (blue). (F) Diagram of the AurKA gene structure (adapted from GenBank, accession no. AL121914) and AurKA-luciferase reporter pGL1486 (adapted from Tanaka et al²⁴ ). Red lines depict potential TCF4 binding sites, untranslated regions (open box), solid box (open reading frame). (G) Cells transfected with pGL1486 and increasing β-catenin amounts assayed similar to panel A show increased AurKA-luciferase activity. (H) Chromatin IP (ChIP) assays in MCF-7 cells demonstrated direct binding of β-catenin transcriptional complex to AurKA gene promoter sequences. The bar graph represents ChIP/immunoglobulin G (IgG) signal normalized to DNA concentration. A nonspecific primer (Neg1) and water were used as negative controls; known primers against the TBE1 region of the c-Myc promoter (c-Myc) was used as a positive control; AurKA primer pairs A and B were designed around regions of putative TCF4 binding sites of the AurKA promoter, described in panel F (bottom). The average fold difference in relative enrichment (ChIP/IgG) over the negative control is represented.