Figure 5.

Interaction of Mss4 with eIF3F prevents the eIF3F-mediated apoptosis. (a) To measure the interaction between different Mss4 mutants and eIF3f in a direct yeast-binding assay, yeast Y190 cells were transformed with GAL4 DNA-binding domain (BD) and GAL-4 activation domain (AD) chimeric constructs as indicated and a β-Gal filter assay was performed as described in Materials and Methods. (b) HEK 293 cells were transiently transfected with DNA constructs as indicated, left untreated (lanes 1–3) or stimulated with 5 mM H₂O₂ for 8h (lanes 4–6) and lysed with TLB. The presence of myc-tagged eIF3f protein in GST–Mss4 wt or GST-Mss4 F75A pull downs (PD) were detected by immunoblotting (IB) with myc antibody (top blot). To ascertain equal amounts of precipitated Mss4 or GST proteins, the blot was redeveloped with antibodies against GST. The blot on the bottom shows the expression in transfected cells of eIF3f protein. 10 μg of total cell lysates were loaded. (c) A7 melanoma cell stably overexpressing Mss4 wt or Mss4 F75A were stressed with 2.5 or 5 mM H₂O₂ for 16h and phase-contrast pictures were taken to document morphological changes. (d) A7 melanoma cell stably overexpressing Mss4 wt or Mss4 F75A were transfected with ctr or Mss4 specific siRNA and stimulated 48h later with 5 mM H₂O₂ for additional 8h. Numbers over the columns represent the amount of apoptotic cells that were determined by flow cytometry after staining of cells with PI. Mean values±S.D. from three repeated experiments are shown. **P<0.01 and ***P<0.001 relative to vector-transfected control cells, t-test. (e) A7 melanoma cells were transiently transfected with GST-tagged Mss4 and myc-tagged eIF3f, either separately or together and stressed with 5 mM H₂O₂ for 8h. Phase-contrast images were taken to document morphological changes in stressed (lower panels) compared with control (upper panels) cells. (f) Western blot analysis of cells examined in (g) demonstrating different expression levels of endogenous and recombinant Mss4 proteins. β-actin immunoblot served as loading control. (g) To stably overexpress the Mss4 protein, A7 melanoma cell were retrovirally transduced with myc-tagged wt or F75A mutated Mss4 variants. Vector-transduced cells served as control. To stably downregulate the endogenous Mss4, A7 cells were infected with lentiviruses containing either scrambled or Mss4 specific shRNA. The five A7 cell lines with different amounts of endogenous or recombinant Mss4 proteins were transfected with myc-tagged eIF3f or empty vector and 40 h later stimulated with 5 mM H₂O₂ for further 8h. The amount of apoptotic cells was documented by flow cytometry after PI staining. Mean values±S.D. from two repeated experiments are shown. *P<0.05, **P<0.01 and ***P<0.001 relative to control vector-transfected cells, t-test