Fig. 9. Translational repression of tethered Ago2 required endogenous TNGW1 and/or GW182. (A) The repression effect of tethered Ago2 was abolished when TNGW1 and GW182 were knocked down, whereas tethered TNGW1 or GW182 maintained repression in the absence of Ago2. HeLa cells were transfected with different siRNA 24 hours prior to the transfection of NHA-tagged constructs and RL-5BoxB/FL reporters. The translation-repression effect related to tethered Ago2, TNGW1 or GW182 was determined by reduction of RL activity and the repression effect in siRNA to EGFP (siGFP) transfected group was normalized to 1. The repression effect caused by NHA-Ago2 was abolished when cells were treated with siGW182 (asterisk, P<0.01, t-test). Knocked down of Ago2 (siAgo2) caused no significant effect on tethered GW182 or TNGW1 induced translational repression. Error bars represent standard deviations. (B) Tethered Ago2-mediated gene silencing required TNGW1 and/or GW182 regardless whether FL-5BoxB or RL-5BoxB was used as targeted reporter. Similar experiments were performed as described previously except for the use of RL-5BoxB reporter together with FL reporter as co-transfection quantitative control. The repression effect of tethered Ago2 was abolished consistently in the abscence of TNGW1 and/or GW182 when FL-5BoxB or RL-5BoxB were used as reporters, whereas tethered GW182 maintained its repression to both reporters in the absence of Ago2.