Human Ago proteins play important roles in miRNA-mediated gene silencing. ECV304 cells were transfected with 100 nM asON or siAgo, then 24 h later with 100 nM mlet-7a, RL-Hmga2m7, and pGL3-control. The day after cells were collected, the Renilla luciferase (RL) and firefly luciferase (FF) activities were measured, and the ratio RL/FF was built. Every experiment was performed in triplicate and values were normalized to asON_Ctr or siscr, which were set at 1. Data were then compared using Friedman test and the Program Prism 4 (GraphPad Software) (n = 9–15). Indicated are mean values ±SEM; *P < 0.05, **P < 0.01, ***P < 0.001. (A,B) Effect of single hAgo depletion via siAgo (A) or asON (B) on the miRNA-mediated silencing pathway. (C,D) Effect of cosuppression of two hAgos on the miRNA-mediated silencing pathway. ECV304 cells were transfected with 100 nM each siAgos (C) or asON (D) (asON_Ctr or siscr in a concentration of 200 nM), then 24 h later with mlet-7a, RL-Hmga2m7, and pGL3-control as described. (E,F) Effect of down-regulation of three hAgos on the miRNA-mediated silencing pathway. ECV304 cells were transfected with 100 nM each siAgo (E) or asON (F) (asON_Ctr or siscr in a concentration of 300 nM).
