Fig 2: FADD phosphorylation during mitotic HeLa.S-Fucci cells. Cells were arrested at the border of G1 and S phases with a double-thymidine block, followed by release to enter S phase. Cell lysates from cells at each time point were subjected to SDS–PAGE, followed by immunoblotting with antibodies as indicated. Cyclin B and cyclin E were used for cell cycle markers of G2/M and G1/S phases, respectively

Fig 3: Isolation of DUSP26 as a FADD phosphatase and an AK2-binding partner.(a) HEK293T cells were co-transfected with p-FADD-HA and each phosphatase cDNA for 24 h, cell extracts were subjected to western blotting using anti-p-FADD and anti-FADD antibodies and to phosphatase assays with pNPP. Bars represent mean±s.d. (n=3). (b) HEK293T cells were co-transfected with FADD-HA and pcDNA3, Flag-DUSP26 or Flag-DUSP26 C152S for 24 h, and cell extracts were then analysed by western blotting. (c) HeLa cells were transfected with pSuper, pDUSP26 or pDUSP26 shRNA for 36 h, after which cell extracts were subjected to western blotting using anti-p-FADD, anti-FADD and anti-α-tubulin antibodies. Total RNAs were purified and analysed with RT–PCR using DUSP26- or GAPDH-specific synthetic oligonucleotides as primers. (d) HEK293T cells were transfected with p3 × Flag or p3 × Flag-DUSP26 for 36 h, and cell extracts were prepared and subjected to the pull-down assay using anti-FLAG agarose-beads. The bound proteins were resolved by SDS–PAGE, stained with Coomassie-blue, and analysed by LC-MS/MS or analysed by western blotting using the indicated antibodies. NS indicates non-specific signal. (e) HEK293T cells were co-transfected with pAK2-HA and pFlag-DUSP26 for 36 h and cell extracts were subjected to immunoprecipitation (IP) assay using anti-HA or anti-Flag antibody. Whole-cell lysates and the immunoprecipitates were probed by western blotting with anti-FLAG or anti-HA antibody. (f) HeLa cell extracts were subjected to IP analysis using anti-AK2 or anti-DUSP26 antibodies and then the immunoprecipitates were analysed by western blotting with the indicated antibodies. (g) AK2 over-expression enhances the binding of DUSP26 to FADD. HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either GFP or AK2-GFP for 36 h, after which cells extracts were subjected to IP analysis using anti-HA antibody. Expression levels of Flag-DUSP26, FADD-HA, GFP and AK2-GFP in whole-cell lysates were examined by western blotting with the indicated antibodies. (h) HEK293T cells were co-transfected with Flag-DUSP26, FADD-HA and either pSuper or AK2 shRNA for 36 h. Cell lysates were subjected to IP analysis with anti-HA antibody, and expression levels of Flag-DUSP26, FADD-HA and AK2 in whole-cell lysates were examined by western blotting.

Fig 4: AK2 deficiency is detected in tumour cells with alteration in cell proliferation.(a) AK2 expression is downregulated in MCF-7 and C33A tumour cells showing increased p-FADD. The expression profiles of AK2, FADD and p-FADD were examined in cancer cells by western blot analysis. Relative ratios (fold) of AK2 (open box) or p-FADD signal (filled box) to FADD on the blots were determined by densitometric analysis. (b) AK2 expression is downregulated in breast tumour cells showing increased level of p-FADD. The expression profiles of indicated proteins were examined in breast cancer cells by western blot analysis. (c) Reconstitution of C33A and MCF-7 cells with AK2 reduces FADD phosphorylation. C33A and MCF-7 cells were stably transfected with pcDNA3 (C33A/Control and MCF-7/Control) or AK2 (C33A/AK2 and MCF-7/AK2). Cell extracts were prepared and subjected to western blotting with the indicated antibodies. (d) Reconstitution of AK2 results in retardation of cell proliferation in C33A and MCF-7 tumour cells. Cell proliferation of C33A/Control, C33A/AK2, MCF-7/Control, and MCF-7/AK2 stable cells were examined for 6 days. Values are the mean±s.d. (n=3). P<0.01; t-test. (e) AK2 suppresses cell proliferation in wild-type MEFs but not in FADD−/− MEFs. FADD+/+ and FADD−/− MEFs (7 × 103) were transfected with either pcDNA (Cont) or pAK2-HA and the cell numbers were counted at different time points. Values are the mean±s.d. (n=3). P<0.01; t-test. (f) Functional dissection of AK2 domains responsible for FADD dephosphorylation and cell growth inhibition. A schematic diagram of the AK2 deletion mutants and a summary of the FADD dephosphorylation and the cell growth inhibition are described. All of AK2 constructs were fused with GFP.

Fig 5: TY (5 μg/mL) induced the expression of the death factors/receptors Fas L/Fas and TRAIL/DR5. (A) The expression levels of FAS mRNA at 48 h after TY treatment estimated by qRT-PCR (left figure). Time-dependent protein expression of p-FADD and death factors/receptors Fas-L/Fas and TRAIL/DR5 were examined by Western blot analysis. (B) Time-dependent expression of MIR34a after treatment with TY estimated by qRT-PCR. Expression levels of MIR34a are shown as the relative ratios with respect to the RNU6B expression levels. (C) Ectopic expression of MIR34a inhibited cell growth dose-dependently and induced apoptosis according to Hoechst 33348 staining at 72 h after transfection. The apoptotic cells are indicated by white arrows. (D) Expression of MIR34a-targeting E2F3, SIRT1, and CyclinD1 was downregulated by the TY. (E) Anti-MIR34a (Anti; 10 and 20 nM) partly canceled the activation of caspase-8 and PARP cleavage elicited by co-transfection with MIR34a. (F) Silencing of E2F3 by siR-E2F3 or MIR32a increased in the expression levels of DR5, TRAIL, and Fas.