Figure 3.

Effects of Ago and Dicer protein knock-downs on expression of RL reporters. (A) Activity of RL reporter constructs in cells with knock-downs of individual Ago proteins (for Ago1-kd and Ago4-kd, lines #1 were used). Luciferase assays were performed 2 days after transfection (60 h post-induction with Tet). Histogram shows normalized mean values (±SEM) of RL/FL activity from three different experiments performed in duplicates (except for Ago3-kd done in two duplicate experiments). (B) Northern blot analysis of expression of RL reporters in Ago2-kd and Ago3-kd lines. mRNA was isolated from cells transfected and induced as described in (A). RL reporter mRNA expression was normalized to EGFP mRNA expressed from a co-transfected plasmid. Histogram shows normalized mean values of RL mRNAs relative to RL mRNA level in cells transfected with the control RL reporter (pRL-Con), which was set to 100%. Error bars (SEM) are derived from four northern blot experiments. Northern blot phosphorimager scans below the graph show results of one representative experiment. (C) Effect of Dicer knock-down on activity of RL reporters. Dicer-kd 2b2 cells were induced with Dox and harvested 3 and 7 days post-induction. Transfection with reporter constructs was performed 2 days before collection. Histogram shows normalized mean values (±SEM) of RL/FL activity from three different experiments performed in duplicates. Small repression of RL-Perf (but not of RL-3xBulgeA and RL-3xBulgeB reporters) still observed even at day 7 likely reflects activity of the residual RISC complex, which turns over when executing RNAi but is required in stochiometric amounts when mediating translational repression.