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. 2018 Oct 9;18:15–24. doi: 10.1016/j.molmet.2018.10.003

Figure 3.

Figure 3

Loss of Ago2 abolishes miRNA-mediated targeting of AMPKα. A, Quantitative real-time PCR analysis of AMPKα1 after immunopreciptation of Ago2 from liver tissue of C57BL/6 mice either fasted overnight for 16 h (white bar) or at random-fed (black bar) (n = 3–5). B, Western blotting analysis of Ago2, AMPKα and phosphorylated AMPKα at Thr172 (p-AMPKα) from liver of 8-week-old Alb-Cre, Ago2flox/flox mice and littermate controls after overnight fasting (n = 3). C, qRT-PCR analysis of microRNAs from liver of 10-week-old Alb-Cre, Ago2flox/flox mice (n = 7) and littermate controls after overnight fasting (n = 6). D, Luciferase assays in HEK cells testing direct targeting of AMPKα genes by miR-148a (148a-mimic), miR-27b (27b-mimic), miR-124 (124-mimic), miR-128 (128-mimic). E, F, Western blotting and qRT-PCR analysis of AMPKα after transfection of either a miR-148a mimic or a pool of control mimics into mouse primary hepatocytes. G, Western blotting analysis of Ago2, AMPKα and phosphorylated AMPKα at Thr172 (p-AMPKα) (n = 3) and qRT-PCR of Ago2 and AMPKα1 from the liver of 10-week-old WT mice during random-fed and overnight fasted conditions (16 h) (n = 5). H, Western blotting analysis of Ago2, AMPKα and phosphorylated AMPKα at Thr172 (p-AMPKα) (n = 3) and qRT-PCR of Ago2 and AMPKα1 from the liver of 10-week-old Lepob/ob mice and wild-type (WT) littermates during random-fed and overnight fasted conditions (n = 6). I, Western blotting analysis of phosphorylated acetyl-CoA carboxylase at Ser79 (p-ACC), total ACC and fatty acid synthase (FAS) from liver of 8-week-old Alb-Cre, Ago2flox/flox mice and littermate controls after overnight fasting (n = 3). J, Quantification of total liver mass of Alb-Cre, Ago2flox/flox mice (n = 4) and littermate controls (n = 6). K, Quantification of liver triglyceride content of Alb-Cre, Ago2flox/flox mice (n = 7) and littermate controls after overnight fasting (n = 7). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.