FIG 4.

Downregulation of DICER1 by hAgo2 is not through miRNAs. (A) Western blot of various proteins involved in the miRNA pathway using samples from HeLa cells transfected with miRNA Let-7a. (B, left) Western blot of DICER1 and hAgo2 proteins using samples from HeLa cells stably transfected with control sponge (labeled as CXCR4) or sponges to remove Let-7a or Let-7b. (Right) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with different sponges and transiently transfected with the plasmid expressing hAgo2. (C) Renilla luciferase activity was measured using samples from HeLa cells with different amounts of pIS1-DICER1 long-mut-let7 (pIS0 empty firefly luciferase plasmid as control). (D, left) Western blotting of DICER1 and hAgo2 proteins using samples from HeLa cells stably transfected with sponges to remove miR103/107. (Right) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with miR103/107 sponges and transiently transfected with the plasmid expressing hAgo2. (E) Luciferase activity was measured using samples from HeLa cells with pIS1-DICER1 long-mut-miR103/107. (F) Renilla luciferase activity was measured using samples from HeLa cells with the pIS1-DICER1 long ALL mutant. (G) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with shRNA targeting GW182 (two different shRNA clones) and transiently transfected with expressing plasmids for hAgo2. Results in panels C, E, and F are the means (±SD) from three independent experiments.