NCL, through the regulation of a subset of miRNAs, modulates cancer cell proliferation in vivo. (A) MDA-MB-231 cells were injected subcutaneously into the flanks of nude mice. 1 wk after injection, mice with comparable tumor sizes were selected (n = 8 for each group of treatment). Mice were treated with intratumoral injections of si-Scr, si-NCL + miRNAs, si-NCL, or si-NCL + Scr. The graph represents the mean tumor volume (mm³) at the indicated days during the experiment for the four groups. Values represent the mean ± SD. (B) Representative images of hematoxylin and eosin (H&E) and IHC staining for Ki-67, NCL, PTEN, pAKT, and active Caspase-3 in xenograft tumors for each treatment group (si-NCL ± Scr groups were unified). (C) Quantitative analysis of IHC stainings reported in B (n = 8). Reported data represent the mean ± SD from three independent slides stained in triplicate. (D) Representative images of immunoblot analysis of total protein extracts from xenograft tumors obtained from the four different groups of treatment using anti-NCL and anti-DICER antibodies (left). GAPDH was used as internal loading control, and densitometric analysis ± SD is also reported (n = 8; right). (A–D) All data reported are representative of three independent experiments. (E) Evaluation by qRT-PCR of mature miR-21, miR-103, miR-222, miR-221, and miR-30a expression levels in subcutaneous xenograft tumors from MDA-MB-231 cells after si-NCL or si-Scr treatment as described in A. Each data sample was normalized for the endogenous RNU48 by the 2^−Δct method. Horizontal lines indicate the median.