AS1411, through posttranscriptional regulation of NCL-targeted miRNAs, affects gene expression in breast cancer cells. (A) miRNA (left) and pri-miRNA (right) expression levels from MCF-7 cells treated with 10 µM AS1411 or its control drug (DCT) at different time points (24, 48, 72, and 96 h) were analyzed by qRT-PCR. For miRNA and pri-miRNA analysis, values were normalized to DCT at the same treatment time. The experiments were performed in triplicate and represent the mean ± SD. The red line represents a trend toward miRNA down-regulation upon AS1411 treatment. (B) Immunoblot analysis of either cytoplasmic (Cyt) or nuclear (N) extracts from MCF-7 cells using antibodies directed to the indicated proteins in the presence of control (DCT) or AS1411 treatment. LAMIN-B1, HSP90, and DROSHA were used as internal control for nucleocytoplasmic separation. Reported data represent three independent experiments. (C) Immunoprecipitation (IP) experiments on HeLa cells transfected with Flag-DGCR8 expression vector and treated with 10 µM AS1411 or control (DCT) for 24 h. Total cell lysates were immunoprecipitated using anti-Flag or control IgG antibodies and analyzed by immunoblot using the indicated antibodies. Inputs are also reported. (D) qRT-PCR analysis of the indicated genes on MCF-7 cells after AS1411 at different time points. *, P < 0.05; **, P < 0.01, relative to DCT-treated cells. Reported data represent the mean ± SD from three independent experiments in triplicate.