Figure 5

TIAF1 self-association induces expression of Smad4 and WOX1. (a) MCF7 cells were transiently transfected with ECFP and EYFP (C/Y) or ECFP-TIAF1 and EYFP-TIAF1 (Tc/Ty) by liposome. The cells were treated with or without TGF-β1 (5 ng/ml) for 24 h. TIAF1 self-binding was analyzed by FRET microscopy.^{11, 17, 35} FRETc shows the extent of protein/protein binding.^{11, 17, 35} TIAF1 self-binding led to Smad4 expression, as determined by immunofluorescence staining. Statistical analysis: all tests versus C/Y controls; Student’s t-test (n=6). The induced Smad4 colocalized with TIAF1 in the cytoplasm and the spiny protrusion ( × 400 magnification; Supplementary Figure S10). (b) Transiently overexpressed EGFP-TIAF1 upregulates the expression of Smad4 and WOX1 in MCF7 (non-reducing SDS-PAGE) and other types of cancer cells (data not shown). Exposure of cells to Prima-1 (10 μM) for 1 h to activate p53 resulted in increased polymerization of TIAF1 and WOX1, but not Smad4. (c) COS7 cells were transiently overexpressed with EGFP-TIAF1 or EGFP. Significantly increased expression of WOX1 is shown, where these proteins colocalize with TIAF1 aggregates, as determined using specific antibodies (n=10; mean±S.D.; experiments versus EGFP controls, Student’s t-test). Dominant-negative TIAF1 (E22/23A) and EGFP failed to induce the indicated protein expression. (d) To stimulate endogenous TIAF1 aggregate formation, COS7 and/or SK-N-SH cells were co-cultured on the ECM of prostate DU145 cells for48 h. Aggregate formation of endogenous TIAF1, along with WOX1 expression, is shown in the cytoplasm of both cells (top two rows). No TIAF1 aggregate formation was observed by culturing COS7 cells alone on the ECM (third row from the top). R48-2 blocking peptide abolished the immunofluorescence (bottom row). (e) NCI-H1299 cells were transiently overexpressed with ECFP-Smad4 and EYFP-TIAF1, followed by treating with TGF-β1 (5 ng/ml) for 24 h. Increased binding of Smad4 and TIAF1 positively correlates with upregulation of APP and Aβ