(A) Western blot assessing suppression of Argonuate-2 (Ago2) in NIH/3T3 and eIF4AIIem1JP cells. Cells were transduced with lentivirus expressing an shRNA targeting Ago2 (TRCN0000255786) or shGFP (SHC002) and extracts were probed with the indicated antibodies by Western blotting. (B) Suppression of gene expression by siRNAs is unaffected by loss of eIF4AII. (Top panel) Schematic representation of the Renilla Luciferase (RLuc) reporter harboring six siCXCR4 binding sites. (Bottom panel) Bar graph reporting on expression levels obtained from the indicated cells transfected with RL6X and pcDNA3-HCV-FLuc in the presence of a scrambled siRNA control (siScr: Dharmacon's ON-TARGETplus Non-targeting siRNA #1) or CXCR4 siRNA (5′-UGUUAGCUGGAGUGAAAAC-3′) (Wang et al. 2009). Luciferase activities were measured 48 h post-transfection. The fold siCXCR4 repression was determined by normalizing RLuc/FLuc values to the values obtained in the presence of the scramble siRNA control. N = 3 biological replications, with each experiment performed with technical triplicates ± SEM. (*) P < 0.05; n.s., P > 0.9. (C) Schematic representation of reporters used to assess miRNA activity. (D) Let7 miRNA-mediated repression is unaffected by loss of eIF4AII. Bar graph reporting on expression levels obtained from the indicated cells transfected with pRL reporters and HCV/FLuc. Luciferase activities were quantitated 48 h post-transfection and plotted with respect to values obtained with the pRL control vector. N = 3 biological replicates (performed in technical triplicates) ± SEM. (**) P < 0.01; n.s., P > 0.15.
