Fig. 5

Transiently overexpressed WWOX/TIAF1/p53 triad induces apoptosis, and determination of the kinetics of the triad formation. a L929 cells were electroporated with indicated plasmids. Overexpression of WWOX, TIAF1 and p53 induced apoptosis. Dominant-negative p53 (S46G) suppressed apoptosis mediated by p53 and TIAF1. EP: electroporation with medium only (Mean ± S.D., n = 3, *p < 0.05, student’s t test). b Ras rescue-based yeast two-hybrid analysis revealed the binding of TIAF1 to the N-terminal first WW domain of WWOX, as shown by the growth of yeast at 37 °C [29,30,31]. WWOX(Y33R) mutant abolished the binding. The SDR domain failed to bind TIAF1. In positive binding controls, MafB self-association and WWOX/p53 binding are shown, and empty pSos versus empty pMyr as negative controls. cDNAs constructed in the pSos are shown in the left, and pMyr in the right. c TNFα, at 100 ng/ml, rapidly upregulated the expression of TIAF1 in 30 min in L929 cells. Co-immunoprecipitation showed the binding of TIAF1 with WWOX. d By co-immunoprecipitation, TGF-β1 (5 ng/ml) had little or no effect on the binding of TIAF1 with pY-WWOX during treatment for 30 min in MCF7 cells. IgH: IgG heavy chain. e, f NCI-H1299 cells were transiently transfected with p53-DsRed, TIAF1-EGFP, and WWOX-ECFP. By 3-protein FRET analysis [37, 39], Prima-1 induced p53 activation-dependent binding with WWOX and TIAF1 with time (see the number in red under FRETc columns, e Experiments were designed to let the FRET energy transfer from WWOX-ECFP to TIAF1-EGFP, and finally to p53-DsRed [37, 39]. Prima-1 increased the triad complex formation in COS7 cells (f). g In Mv1Lu cells, TGF-β1 rapidly increased the triad complex formation in 1 h, followed by reduction (n = 6; Student’s t test; experiments versus time 0 controls). h Prima-1 increased the binding of the first WW domain of WWOX with TIAF1 and p53 with time. Each bar represents an average of two experiments in (f) and (h)