Fig 2: TIAF1 aggregates in degenerative neurons in the AD hippocampus. (a) Human hippocampal tissue sections were pre-stained with Bielschowski stain (containing silver), followed by staining with specific antibody against TIAF1. Fluoro-Jade C Red was used to identify degenerative neurons. Nuclei were stained with DAPI. Sequential magnification of aggregated TIAF1 (green; from × 40 to × 400) shows its colocalization with degenerative neurons (both in red and blue). (b) TIAF1 aggregation is shown in the extracellular matrix containing no signal for nuclei. (c) A representative negative control. The above sections were stained with a secondary antibody (green), Fluoro-Jade C Red and DAPI. Scale bars a–c, 20 μm for × 400 magnification, and 200 μm for × 40 magnification. (d) A confocal image of a plaque, containing TIAF1 and Aβ, is shown ( × 600; also see data in Supplementary Figure S10). Scale bar, 20 μm for × 600 magnification. (e) A confocal image of a TIAF1 plaque, containing de novo synthesized Aβ aggregates in the epicenter, is shown from the hippocampal section of an APP/PS1 transgenic mouse (also see Supplementary Figure S11)

Fig 3: 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

Fig 4: TIAF1 aggregation in vivo and generation of Aβ in vitro. (a) Water-soluble TIAF1 protein aggregates were analyzed by non-reducing SDS-PAGE (5%) and western blots using the lysates of hippocampal samples from postmortem AD patients and nondemented controls. The blots were stained with specific anti-TIAF1 antibody (also see Supplementary Figure S13). C, control; A, Alzheimer's disease. (b) Filter retardation assay shows the presence of ‘water-insoluble' TIAF1 aggregates in control and AD hippocampi (representative data from 138 samples). The amounts of protein loaded were 10, 30 and 60 μg, respectively, from top to bottom. (c) TIAF1 aggregation is shown in 59% of nondemented control hippocampi (age 59.0±17.0, n=41), and only 15% of the total samples have Aβ aggregates. In comparison, 54% of TIAF1 aggregation is shown in the hippocampi of older postmortem AD patients (80.0±8.8, n=97), and 48% of the total AD samples possess Aβ aggregates. In addition, there was 17% of the total TIAF1-positive samples possessing Aβ aggregates in the control group, and 48% in the AD group. (d) L929 cells were treated with TNF-α (50 ng/ml) for 24 h. Presence of Aβ monomer (∼4.5 kDa) is weakly shown in the whole cell lysate (see load), and the band was enriched by immunoprecipitation using anti-Aβ antibody (AβH43). (e and f) Transiently overexpressed ECFP-TIAF1/EYFP-TIAF1 (Tc/Ty) induced generation of amyloid fibrils (dot blot; AO antibody), AICD and Aβ (reducing SDS-PAGE), but not in cells overexpressing ECFP/EYFP (C/Y) or ECFP-TIAF1/mutant EYFP-TIAF1 (Tc/mTy). TGF-β1 (5 ng/ml) induced the expression of prefibril Aβ oligomers (A11 antibody) in C/Y-expressing L929 cells. Mutant or dominant-negative TIAF1 is with an E22/23A alteration. (g) Similar experiments were performed in Mv1Lu cells, which shows Tc/Ty-induced amyloid fibrils (a single band under reducing SDS-PAGE; ∼60 kDa). (h and i) NCI-H1299, B16F10 and SH-SY5Y cells were treated with medium C, TGF-β1 (5 ng/ml), -β2 (5 ng/ml), -β3 (5 ng/ml), TNF-α (50 ng/ml) and TNF-β (25 ng/ml) for 24 h. Distinct APP degradation patterns are shown. A representative protein gel is shown from three repeats e–i

Fig 5: TGF-β1 induces TIAF1 self-association, suppression of APP phosphorylation at Thr668, and Aβ superinduction. (a and b) SK-N-SH cells, overexpressing ECFP/EYFP or ECFP-TIAF1/EYFP-TIAF1, were treated with TGF-β1 (5 ng/ml) for 24 h, followed by determining the extent of protein self-binding by FRET microscopy. The expression of APP, APP phosphorylation at Thr668 and Aβ was determined by fluorescent immunostaining. (c) FRET analysis was carried out from 10 assays (mean±S.D., n=10, Student's t test: experiments versus non-treated C/Y controls). C/Y, ECFP/EYFP; Tc/Ty, ECFP-TIAF1/EYFP-TIAF1. (d) Comparison of protein expression profiles is shown (mean±S.D., n=50, Student's t test: experiments versus non-treated C/Y controls). (e) TGF-β1 increased the phosphorylation of endogenous APP at Thr668 in MCF7 cells. By immunoprecipitation using specific TIAF1 antibody, TGF-β1 suppressed the binding of TIAF1 with phosphorylated APP (∼70 kDa), but did not affect the binding of TIAF1 with Aβ monomer. (f) TGF-β2 increased the binding of TIAF1 with amyloid fibrils in L929 cells during treatment for 24 h. IgH, IgG heavy chain. (g) In comparison, exposure of B16F10 cells to TGF-β1 for 1 h caused reduction in the binding of TIAF1 with amyloid fibrils, whereas the effect was barely visible during this short-term experiment for HCT116 cells. L929, B16F10 and HCT116 were grown up to 100% confluence