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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 2: — PB integrity is indispensable for ARE-mRNA degradation induced by TTP. (A) Hedls knockdown completely destroyed the PB integrity. HeLa cells transfected with control siRNA (siNC) or siHedls-1 or siHedls-2 were stained with anti-Dcp1a antibody to visualize PBs. (B) PB depletion by Hedls knockdown increases FL-GM-CSF reporter mRNA level mediated by TTP. 293T cells transfected with control siRNA (siNC) or siHedls-1 or siHedls-2 were also transfected with FL-GM-CSF reporter plasmid and overexpression plasmids HA-tagged TTP or Septin1. The normalized values of FL mRNA level were set to 1 for cells transfected with Septin1 in each knockdown condition. Means and SD from three independent experiments are shown. *p < 0.05; **p < 0.01. (C) Results of an experiment similar to that for B, except that the reporter FL-GM-CSF was replaced with FL-TNF. *p < 0.05. (D) Lsm1 knockdown destroyed the PB integrity. HeLa cells transfected with control siRNA (siNC) or siLsm1 were stained with anti-Dcp1a antibody to visualize PBs. (E) PB depletion by Lsm1 knockdown increases FL-GM-CSF reporter mRNA level induced by TTP. 293T cells transfected with control siRNA (siNC) or siLsm1 were also transfected with FL-GM-CSF reporter plasmid and overexpression plasmids. The normalized values of FL mRNA level were set to 1 for cells transfected with Septin1 in each knockdown condition. *p < 0.05. (F) Results of an experiment similar to that for E, except that the reporter FL-GM-CSF was replaced with FL-TNF. *p < 0.05. (G) RCK silencing diminished the cytoplasm PB foci as determined by anti-Dcp1a staining. (H) The experiment was the same as for E, except that the siLsm1 was replaced with siRCK. *p < 0.05. (I) EIF4E-T knockdown destroyed the PB integrity as shown by anti-Dcp1a staining. (J) The experiment was the same as for B, except that the siHedls-1 and siHedls-2 was replaced with sieIF4E-T-1 and sieIF4E-T-2. *p < 0.05. (K) Hedls knockdown increased ARE-mRNA stability induced by TTP. HeLa cells were transfected with control siRNA (siNC) or siHedls-1 twice. In the second transfection, β-GM-CSF reporter plasmid, HA-TTP or HA-Septin1, and the internal control plasmid FL-GAPDH were also included in the transfection mixture in the presence of tetracycline (50 ng/ml). Pulse-chase experiments were performed as described under Material and Methods. The normalized values of β-globin mRNA level were set to 100% for cells transfected with TTP compared with cells transfected with Septin1, at the 0 time point, in each knockdown condition. Means and SD from three independent experiments are shown. (L) The experiment was the same as for K, except that the siHedls-1 was replaced with sieIF4E-T-1.