FIG. 3.

Effects of knockdown or overexpression of hUPF1 on endogenous miRNA targets. (A) qRT-PCR shows that the targets of endogenous miRNAs are upregulated when hUPF1 or hAGO2 was knocked down in HeLa cells (n = 3; means ± standard deviations [SD]). RT-PCR (lanes 1 to 5) was carried out to confirm the knockdown of hUPF1 and hAGO2. (B) Cells were prepared as for panel A and were used for Western blotting. The graph shows the normalized levels of the p27 protein from three independent experiments (n = 3, means ± SD). α, anti. (C, left) qRT-PCR shows that the overexpression of hUPF1 decreases miRNA targets. A Flag-tagged wild-type hUPF1 (WT) or mutant hUPF1 (R844C) expression plasmid was transfected into HeLa cells, and the target mRNA levels were determined by qRT-PCR (n = 3; means ± SD). A paired one-tailed t test was used to calculate the P values for qRT-PCR (****, P < 0.001; ***, P < 0.01; *, P < 0.1). (Right) Expression levels of the Flag-hUPF1 proteins were measured by Western blotting.