Figure 6

TIAF1 is essential for apoptosis mediated by WOX1, p53 and dominant-negative JNK1. (a) Mv1Lu cells were transfected with expression plasmid constructs of WOX1 and/or TIAF1si by electroporation, followed by culturing for 48 h. The extent of cell growth was measured by MTS proliferation assay. TIAF1 knockdown cells resisted WOX1-induced growth inhibition (n=8; mean±S.D.; Student’s t-test: experiments versus scramble controls). Scram=‘scrambled RNA’ control plasmid. (b) L929 cells were transfected with WOX1 and/or TIAF1si plasmids, and then grown in soft agarose for 3 weeks to allow colony formation (measured by MTS proliferation assay) (n=3; Student’s t-test; experiments versus scramble controls). (c) When L929 cells were transfected with WOX1 and TIAF1 plasmids (1.25 μg per 10⁶ cells), both expressed proteins synergistically caused cell death (∼40%, n=8). Dominant-negative WOX1 (dnWOX1)¹⁷ and phospho-WOX1 mutants (Y33R and Y61R)¹⁷ failed to induce cell death, in the absence or presence of TIAF1 (n=8; Student’s t-test: experiments versus scramble controls). (d) L929 stable transfectants, expressing a scramble, TIAF1si or WOX1si construct, were established. Transient overexpression of these cells with an empty vector, p53 or dnJNK1 construct was carried out, and the extent of cell death was measured in 48 h (n=8; Student’s t-test; experiments versus scramble controls). When TIAF1 and WOX1 were knocked down, ectopic p53 and dnJNK1-induced cell death was blocked. (e) In contrast, when TIAF1 was knocked down, Smad4-induced apoptosis of Mv1Lu cells was enhanced (see subG1 phase; a representative data from two experiments). a, SubG1 phase; b, G0/G1 phase; c, S phase; d, G2/M phase