NCL and its target miRNAs are frequently overexpressed in breast cancer. (A) Immunoblot analysis of NCL in total protein extracts from different breast cancer cell lines. Numbers represent the densitometric analysis compared with β-ACTIN as internal loading control. (B) Immunoblot analysis of NCL in total protein extracts from human breast cancer specimens (T) and their normal counterpart (N) that were normalized by total loaded proteins detected by Ponceau staining. “B” indicates benign specimens. (C) Overview of 240 breast samples (57 normal and 183 TNBC) analyzed by NanoString miRNA technology distributed in function of miR-103, miR-222, and miR-21 expression (red, up-regulation; blue, down-regulation). (D) IHC of NCL in three samples of TNBC, which are representative of the different scores assigned to the NCL staining. Insets represent higher magnification (40×) of selected areas. (E) miR-21, miR-103, miR-222, miR-30a, and miR-155 relative expression in NCL-negative, NCL-positive “+”, and NCL-positive “++” samples were determined by NanoString technology. The relative expression values were used to design box and whisker plots. Dots in the boxes indicate outlier points. Kruskal-Wallis analysis assessed that the three miRNAs were differentially expressed among NCL-negative versus NCL-positive “+” samples and NCL-negative versus NCL-positive “++” samples of the Bartlett test (P < 0.001). (F) Representative IHC and miRNA ISH for NCL (red) and miR-221 (left) or miR-21 (right; green) in breast cancer samples. Colocalization (yellow) between NCL and miRNAs was evaluated using the Nuance system. (G) Representative miRNA ISH using a specific miR-21 probe or a nonspecific miRNA scramble (Scr) probe on breast cancer samples.