Figure 2.

Loss of miRNA from Ago2 deactivates miRNPs in LPS-treated mammalian macrophages. (A) Ago2 binding to target mRNA in RAW 264.7 cells expressing a RL let-7a reporter and FLAG-HA-Ago2. Cells were lysed before and after 4 h of LPS treatment and from Ago2 IPed materials RNA was isolated and Ago2-associated RL-3xbulge-let-7a mRNA was quantified (left panel) (***P<0.0002). In similar but separate IP experiments, quantifications of Ago2-associated TNF-α, and IL-6 mRNAs were performed (right panel) (*P<0.0376). (B) A schematic representation of RL-5BoxB reporter used for Ago2-tethering experiments. In tethering assay, RL-5BoxB mRNA was co-expressed with either N-HA-Ago2 or HA-Ago2 and repression level of RL-5BoxB reporter was measured. Association of RL-5BoxB mRNA with N-HA-Ago2 in untreated or 4-h LPS-treated RAW 264.7 cells was quantified. (C) Relative level of active miR-122 RISC present in naïve and 4-h LPS-treated cells were quantified by in vitro RISC cleavage assays (cleaved product is marked by an *). For negative control, miRISC was prepared from naïve RAW 264.7 cells not expressing miR-122. A ³²P 22 nt marker oligonucleotide was loaded as size marker (lane M). (D,E) Change in miRNA binding to Ago2 in LPS-activated macrophages. let-7a level in Ago2 IPed materials isolated before and after 4-h LPS treatment was quantified in RAW 264.7 or mouse primary macrophages isolated from peritoneal exudates (D) or in splenic macrophages of mice intraperitoneally injected with LPS or PBS (E). n=3 for LPS- or PBS-injected mice. (F) Levels of different miRNAs were quantified from HA-Ago2 immunoprecipitates obtained from naïve- or LPS-activated RAW 264.7 cells. (G) Let-7a miRNA association was measured from HA-Ago1 or HA-Ago3 immunoprecipitates before and after LPS treatment. For quantification purpose, mean values±s.d. were determined from three independent assays. LPS, lipopolysaccharide; miRISC, miRNA-induced silencing complex; miRNA, microRNA; miRNP, microRNA protein complex; mRNA, messenger RNA; PBS, phosphate-buffered saline; RISC, RNA-induced silencing complex; RL, Renilla luciferase; TNF-α, tumour necrosis factor alpha.

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