Nestin silencing inhibits the Wnt/β-catenin signaling in breast CSC. Nestin-si and Nestin-c breast CSC were cultured for 48 h, and the relative levels of target proteins and phosphorylation were determined by western blotting. Data are presented as the mean ± SD of the levels of target proteins vs. the β-actin control, or the phosphorylated vs. the total form, from six separate experiments. In addition, Nestinhigh in the presence or absence of LGK974 (1 nM), Nestinlow in the presence or absence of SB216763 (5 μM), and control CSC were tested for their proliferation via mammosphere formation assays. Western blot analysis. (A-C). Quantitative analysis of the relative levels of target proteins. (D) Quantitative analysis of the formed mammospheres. *P <0.05, vs. the Nestin-c or control, except for specifically indicated. CSC, cancer stem cell; SD, standard deviation.