NCL modulates the biogenesis of a subset of miRNAs. (A) Northern blot analysis at 72 h of indicated miRNAs, after either control (si-Scr) transfection or NCL (si-NCL) knockdown in HeLa cells. (B) Mature miRNA expression levels in HeLa cells transfected with the indicated shRNAs were analyzed by qRT-PCR. (C) Mature and pri-miRNA expression levels in HeLa cells transfected with the indicated siRNAs were analyzed by qRT-PCR. (B and C) Values represent the mean ± SD from three independent sets of experiments performed in triplicate. *, P < 0.01. (D) qRT-PCR on HeLa cells after RIP performed using anti-NCL or normal control IgG followed by detection of the indicated pri-miRNAs. GAPDH mRNA was used as internal control. Values were normalized to levels of immunoprecipitated pri-miRNAs using normal control IgG. Immunoprecipitated pri-miRNA levels using normal control IgG were used as normalizer for qRT-PCR experiments. Reported data represent the mean ± SD from three independent sets of experiments performed in triplicate. *, P < 0.01. f.c., fold change. (E) Northern blot analysis of total RNA from HeLa cells cotransfected with pri–miR-21 expression vector (pMIRNA1-pri–miR-21) and with si-NCL, si-DROSHA, and si-Scr, as indicated. RNU6 levels are shown as a loading control. (F) Northern blot analysis of total RNA from HeLa cells cotransfected with pri–miR-221 (left) or pri–miR-155 (right) expression vectors (pMIRNA1-pri–miR-221 and pri–miR-155) and with si-DICER and si-NCL, as indicated. (G) Processing assay of ³²P internally labeled pri–miR-21 incubated with HEK-293 total cell extracts in the presence or absence of 50 nM NCL-³⁵S. (F and G) Images are representative of different experiments at least performed in triplicate.