siRNA-mediated gene silencing in human cells. (A and B) 293 and (C and D) HeLa cells transfected with pEGFP-N3 and antisense (as) ssRNAs and dsRNAs were harvested 48 h after transfection and were assayed for GFP expression by FACS analysis (assayed in triplicate; data are shown as mean ± SEM). A and C show the percentage of GFP-positive cells and B and D show the fluorescence intensity (Geo Mean) of GFP-positive cells. (E–H) HeLa cells transfected with pcDNA3-CAT, ssRNAs, and dsRNAs were harvested 48 h after transfection and assayed for CAT expression (assayed in triplicate and normalized to the amount of CAT pg/μg of protein observed in plasmid plus sense-transfected cells. s, sense ssRNA; as, antisense ssRNA). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

siRNAs and mammalian dsRNA-dependent pathways. To detect PKR autophosphorylation, we performed in vitro kinase assays as described in Methods. (A) In vitro kinase reactions were performed without exogenous RNA (−) or with 1 μg/ml of reovirus dsRNA or 1 μg/ml of siRNA (21–25 nucleotides), or 1 μg/ml of 78- or 81-nt dsRNA. (B) In vitro kinase competition assays were performed by using si- and dsRNAs. Reactions were performed without exogenous RNA (−) or 1 μg/ml of reovirus RNA, or 75-fold excess siRNA (21–25 nucleotides) or 78- or 81-nt dsRNA, plus reovirus dsRNA (1 μg/ml). siRNAs of 21–25 nucleotides and dsRNA of 78 nucleotides corresponded to egfp (the 81-nt dsRNA corresponds to LacZ). (C and D) 293 cells transfected with pEGFP-N3 and unc-22 or egfp siRNAs, and (E and F) 293 cells transfected with pEGFP-N3 and 78 egfp dsRNA and unc-22 or egfp siRNAs were assayed for GFP expression by FACS analysis 48 h after transfection (each transfection was assayed in triplicate; data are shown as mean ± SEM). B and D show the percentage of GFP-positive cells and C and E show the fluorescence intensity (Geo Mean) of GFP-positive cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Gene-specific inhibition of expression in MEFs by siRNAs. MEFs transfected with plasmid DNA, ssRNAs, and dsRNAs were harvested 48 h after transfection and were assayed for (A–D) GFP expression by FACS analysis (each transfection was assayed in triplicate and data are shown as mean ± SEM). A and C show the percentage of GFP-positive cells and B and D show the fluorescence intensity (Geo Mean) of GFP-positive cells. (E–I) CAT expression (each transfection condition was assayed in triplicate; data in E are normalized to the amount of CAT pg/μg of protein observed in pcDNA3.CAT-transfected cells; data in F–I are normalized to the amount of CAT pg/μg of protein in plasmid and sense ssRNA-transfected cells. s, sense ssRNA; as, antisense ssRNA). (J) egfp and neo RNA levels by Northern analysis of poly(A)⁺ mRNA. (K) Cell survival (assayed in duplicate and shown as a mean OD_560–650; dsRNAs of 21–25 and 78 nucleotides correspond to egfp; the dsRNA of 81 nucleotides corresponds to LacZ). (L and M) GFP expression by FACS analysis (data are shown as relative percentage normalized to pEGFP-N3-transfected cells). *, P < 0.05; **, P < 0.01; ***, P < 0.001.