Insulin-like growth factor 1 receptor serves as a marker for breast cancer stem cells. (A) Cells from xenograft BC0145 or BC0244 tumors were sorted as indicated populations and phosphorylation of insulin-like growth factor 1 receptor (IGF-1R) was determined by western blot. (B) pIGF-1RTyr1165/1166 of immunoprecipitated IGF-1R from aldehyde dehydrogenase (ALDH)- or ALDH+ BC0244 xenograft tumor cells was determined. (C) Tumor cells of BC0145 or BC0244 xenografts were stained with PE-conjugated anti-IGF-1R antibody and FITC-conjugated anti-H2Kd antibody. CD24-CD44+ or ALDH+ cells within IGF-1R+/IGF-1R- BC0145 cells (upper panel in (B)) or IGF-1Rhi/IGF-1Rlo BC0244 cells (lower panel in (B)) were determined by co-stain with PE-Cy7-conjugated anti-CD24/APC-conjugated anti-CD44 antibodies or Aldefluor substrate. (D), (E) Two populations of IGF-1R+/IGF-1R- (BC0145) or IGF-1Rhi/IGF-1Rlo (BC0244) cells were sorted from the H2Kd- population by fluorescence-activated cell sorting (FACS) and determined the mammosphere formation capability (D) or tumorigenicity (E). The CSC frequency was calculated by ELDA software (table in (E)). All experiments were repeated independently at least twice and results shown were from a representative experiment. IP, immunoprecipitation; IB, immunoblot.