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. 2018 Apr 15;29(8):988–1002. doi: 10.1091/mbc.E17-02-0105

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© 2018 Qi, Song, et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 4: — The cooperation of TTP and Ago2 is critical for ARE-mRNA degradation. (A) Ago2, acts together with TTP to promote ARE reporter mRNA decay. 293T cells were transfected with the indicated siRNAs and then transfected with reporter FL-GM-CSF or FL-TNF, RL, and the indicated plasmids expressing HA-tagged TTP or HA-Septin1. Normalized value of FL mRNA level was set to 1 for cells transfected with the HA-Septin1 plasmid and the relative value of FL mRNA was shown for cells transfected with the HA-TTP plasmid in each knockdown condition. Means and SD from three independent experiments are shown. **p < 0.01. (B) 293T cells were transfected with the FL-GM-CSF or FL-TNF reporter and RL plasmids, together with two plasmids, one expressing either HA-tagged TTP or HA-Septin1 and another expressing either Myc-tagged Ago2 or Myc-Septin1 or corresponding empty vector, as indicated. The relative value of FL activity was set to 1 for cells transfected with the plasmid expressing HA-tagged Septin1 and the relative value of FL mRNA was shown for cells transfected with the plasmid expressing HA-tagged TTP in each condition. Means and SD from three independent experiments are shown. **p < 0.01; ns, no significance. (C) Ago2 depletion only did not change ARE reporter mRNA level. 293T cells transfected with control siRNA (siNC) or siAgo2 were also transfected with the indicated reporter plasmids. The normalized value of FL mRNA level was set to 1 for cells transfected with siNC for each reporter mRNA. ns, no significance. (D) 293T cells transfected with Myc-Ago2 or corresponding empty vector (control) were also transfected with the indicated reporter plasmids. The normalized value of FL mRNA level was set to 1 for cells transfected with control plasmid for each reporter mRNA. ns, no significance. (E) 293T cells were transfected with the indicated siRNAs and the reporter FL-GM-CSF and RL plasmids, together with two plasmids, one expressing either HA-tagged TTP or HA-Septin1 and another expressing either Myc-tagged Ago2 or Myc-Septin1. The normalized value of FL mRNA level was set to 1 for cells transfected with plasmids HA-Septin1 and Myc-Septin1 in each knockdown condition. Means and SD from three independent experiments are shown. **p < 0.01. (F) Results of an experiment similar to that for E, except that the reporter FL-GM-CSF was replaced with FL-TNF. **p < 0.01.