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. 2014 Jan 27;33(35):4407–4417. doi: 10.1038/onc.2013.594

Figure 5.

Figure 5

Wig-1 interacts with deadenylase subunit CNOT6. Knockdown of the key components of the ARE-mediated mRNA decay pathway CNOT6, DCP1a, PM/Scl-75 and XRN1 was performed in HCT116 p53−/− cells, and the levels of FAS mRNA were assessed through qRT–PCR and compared with levels upon Wig-1 depletion. The figure shows that FAS mRNA levels are increased in a comparable manner following knockdown of CNOT6 or Wig-1 (no significant difference), suggesting that the Wig-1 effect on FAS mRNA is exerted at the level of poly(A) tail deadenylation (a). Protein lysates from HCT116 p53+/+ or HCT116 p53−/− cells transfected with pCMVtag2b or pCMVtag2bhWig-1 were immunoprecipitated with a Flag antibody or CNOT6 antibody cross-linked to Dynabeads Protein G, followed by western blotting with indicated antibodies (b). Coimmunostaining of endogenous Wig-1 (green) and CNOT6 (red) (c) and Wig-1 (green) and SGs marker TIAR (red) (d) in HCT116 p53+/+ cells after 30 min of treatment with 0.5 mM arsenite shows colocalization in cytoplasmic SGs. Arrows indicate the positions of overlapping Wig-1 and CNOT6 foci or Wig-1 and TIAR. Scale bars, 20 μm. DAPI shows DNA staining.