PSG9 expression analysis in Wnt stimulated RKO cells. To determine whether induction of Wnt signaling in cells with wild type APC could induce PSG9 expression, RKO cells with wild type APC and β-catenin were stimulated with either Wnt3a or Kenpaullone (Kenp). After 23 hrs treatment RNA and protein were extracted and processed for PSG9 transcripts expression level by semi-quantitative RT-PCR (a) and β-catenin accumulation by western blot analysis (b). Neither of the treatments nor the RKO-β-cateninS37A (βcat-S37A) stable cell line which expressed constitutively active β-catenin in RKO cells caused expression of PSG9 (a). The SW480 colorectal cancer cell line was used as a positive control (a-b). The RKO cells responded to Wnt stimulation, since cells treated with Wnt3a showed 4-fold induction in luciferase activity compared to untreated cells (c). Axin2, a known downstream target of Wnt signaling, showed 2.4 fold up-regulation in expression as determined by quantitative PCR (d). No PSG9 transcript up-regulation was detected in these cells under these conditions (e). Each sample was analyzed in triplicate.