SOX4 transcriptional activity is modulated by Wnt-induced interaction with plakoglobin. A. ChIP assay of HA-SOX4 bound to the predicted SOX4-binding sites on DICER1, SOX4, DHX9, and AXIN2 promoters. GAPDH is shown as a negative control. B. Quantitative results of ChIP by realtime-PCR. SOX4 promoter occupancy is increased with Wnt treatment, but is strongly reduced by combined WNT3A+LMB treatment. (n = 4; error bars represent SEM; *, p < 0.05 for a one-sided paired t-test) C. SOX4 targets and Wnt downstream genes are inhibited after Wnt induction and LMB treatment. Realtime-PCR expression analysis of SOX4 direct targets and Wnt signaling downstream genes after 24 hrs of WNT3A and LMB treatment. D. siRNA directed against plakoglobin downregulates protein expression in LNCaP HA-SOX4 cell lines. Cells were harvested 48-hr post-transfection and Western blot were probed with anti-plakoglobin and anti-β-actin antibodies in the presence or absence of WNT3A treatment. E. Quantitative ChIP-qPCR assay of HA-SOX4 following plakoglobin knockdown. HA-SOX4 promoter occupancy is strongly reduced by combined WNT3A and LMB treatment but is partially restored when plakoglobin is knocked down by siRNA treatment. Control scrambled siRNA had no effect (n = 3; error bars represent SEM; *, p < 0.05 for a one-sided paired t-test).