RT-PCR shows up-regulation of stem cell markers and only the SP fully repopulates the tumor cell population in vitro. (A) RNA from SP- and non-SP–sorted NXS2 or 4T1 cells were amplified with primers targeting the indicated genes. (B-E) Hoechst 33342 dye–stained 4T1 cells were sorted into 3 separate groups; (B) PI^–/live, (C) SP, and (D) non-SP cells. Following sorting, each group of cells was cultured for 7 days, restained with the Hoechst 33342 dye, and analyzed by FACS to determine the percentage of cells found within each compartment. Each figure represents 1 of 3 experiments. (E) The percentage of repopulation of the non-SP compartment in SP-derived cultures and the percentage of repopulation of the SP compartment in non-SP–derived cultures. Data represent the mean and SEM of the percentage of repopulation.