FIG 3.

hAgo2 downregulates DICER1 expression by affecting the 3′-UTR of its mRNA. (A) Western blot using protein samples from HeLa cells transiently transfected with expression plasmids with or without the MG132 treatment, as indicated. (B) Real-time RT-PCR to detect the DICER1 mRNA amount using RNA samples from HeLa cells transiently transfected with expression plasmids for hAgo2. (C) Real-time RT-PCR to detect the relative mRNA amount of hAgo2 and DICER1 using RNA samples from HeLa cells with shRNA targeting hAgo2 (two different clones, +6 and +8). (D) HeLa cells were transfected with the following plasmids: pGL3-DICER1-prom or pGL3-Basic, 0.1 μg; pRL-TK, 0.01 μg; pcDNA3-myc-hAgo2 (or pcDNA3 empty vector), different doses of 0.1, 0.2, and 0.4 μg. Cells were harvested, and luciferase activity was measured 48 h after transfection. (E) HeLa cells were transfected with the following plasmids: pIS1-DICER1-long UTR or pIS1, 0.1 μg; PIS0, 0.01 μg; pcDNA3-myc-hAgo2 (or pcDNA3 empty vector), different doses of 0.1, 0.2, and 0.4 μg. The cells were harvested, and luciferase activity was measured 48 h after transfection. (F) HeLa cells were transfected with the following plasmids: pIS1-DICER1-long UTR and pcDNA3-myc-hAgo2 (or pcDNA3 empty vector) with different doses of 0, 0.5, 1, and 2 μg. The cells were harvested, and the luciferase mRNA level was determined 48 h after transfection using real-time RT-PCR. Results in panels B, C, D, E, and F are the means (±SD) from three independent experiments.