Nestin silencing inhibits the mammosphere formation, induces cell cycle arrest at G2/M and promotes apoptosis in breast CSC. The isolated CD44+CD24− breast CSC from 26 specimens were transfected with control siRNA, Nestin-specific siRNA, pIRES-Nestin-EGFP, or control pIRES-EGFP to generate Nestin-c, Nestin-si, Nestin+ or Nestin-EGFP cells. The Nestin-c, Nestin-si, and Nestin+ CSC were tested for the levels of Nestin expression by western blotting and for their ability to form mammospheres in vitro. Their cell cycling progression and spontaneous apoptosis were determined by flow cytometry. Data are representative charts and images, and expressed as the mean ± SD of each group (n = 26 per group). (A). Flow cytometric analysis of CD44+CD24− CSC. (B) Western blot analysis of Nestin expression in breast CSC. Nestin-EGFP and Nestin-c control cells showed similar levels of Nestin expression (data not shown). (C) The mammosphere formation. (D) Cell cycle analysis of Nestin-si and Nestin-c breast CSC; (E) apoptotic Nestin-si and Nestin-c breast CSC. *P <0.05 vs. the control. CSC, cancer stem cell; SD, standard deviation; siRNA, small interfering RNA.