Targeting IGF-1R affects cancer stem cell properties and Akt activation of breast cancer stem cells. (A) CD44+ cells of AS-B244 cells were treated with picropodophyllin (PPP) for 12 hours and cell migration within 18 hours was determined (upper panel). E-cadherin expression was determined by immunofluorescence stain (middle panel, 10× objective lens). Cell morphology was observed under microscope (bright field, 20× objective lens). (B) The expression of epithelial-mesenchymal transition-related molecules after PPP treatment was determined by western blot. (C) Cells were sorted as described in Figure 1A and the phosphorylation of Akt or mammalian target of rapamycin (mTOR) was determined by western blot. GAPDH and actin were used as internal control for BC0145 and for BC0244, respectively. (D) AS-B145 or AS-B244 cells were transfected with 100 nM negative control siRNA (ctrl) or insulin-like growth factor 1 receptor (IGF-1R) specific siRNA (KD) for 48 hours and the expression of IGF-1R, Akt or p-Akt was determined by western blot. (E) ALDH+ AS-B145 or ALDH+ AS-B244 cells were treated with PPP for 48 hours. pAktser473was determined by western blot. All experiments were repeated at least twice and results shown were from a representative experiment. ALDH, aldehyde dehydrogenase; DMSO, dimethylsulfoxide.