Figure 5. SSX interacts with β-catenin and transactivates TCF/β-catenin target genes.

A) DFW and Saos-2 cell lines were synchronized in G1/S by double thymidine blockade as indicated in material and methods (0 hrs), and released into normal medium containing FBS for 6 and 24 hrs. SSX was immunoprecipitated from protein extracts collected at the indicated time points using the rabbit anti SSX antibody (FL188, detecting SSX1–9). An equivalent amount of protein from G1/S blocked Saos-2 cells was immunoprecipitated with an irrelevant anti mouse (m) or ant-rabbit (r) antibody. The protein complex were electrophoresed in reducing conditions and blotted ether with goat anti SSX (N18) or mouse anti β-catenin antibodies. The total input levels of β-catenin are shown in the upper gel image. B) Activity of a TCF/Lef luciferase reporter in SSX silenced and control DFW and Saos-2 cells, 48 hours after transfection of siRNA molecules (n = 5). The activity of the TCF/Lef reporter in SSX silenced cells is relative to that of control cells ( = 1). C) Gene transcription associated with SSX expression in both Saos-2 and DFW cells, determinded by PCR arrays containing 84 genes associated with epithelial to mesenchymal transition (n = 5) and confirmed by Q-RT PCR in SSX silenced and control DFW and Saos-2 cells as described in material and methods. SSX was knocked down in Saous-2 or DFW cells using siRNA molecules or shRNA vectors as indicated. Cells were collected and RNA was isolated 6 ours after siRNA transfection or 6 hours after addition of doxycycline into the medium (conditionally shRNA). The loss of SSX expression following RNAi silencing was confirmed by western blot before each Q-RT-PCR array, as shown in the figure. Fold-Change [2∧(−Delta Delta Ct)] is the normalized gene expression [2∧(−Delta Ct)] in SSX silenced cells divided by the normalized gene expression in the control (SSX+) cells. Values less than one indicate a negative or down-regulation.