(A) Representation of the overlap between the ALDEFLUOR⁺ subpopulation and the CXCR1⁺ (top) or CXCR2⁺ (bottom) subpopulation of SUM159 cells. (B and C) SUM159 cells were cultured in adherent conditions and treated with repertaxin and 2 specific blocking antibodies for CXCR1 or CXCR2. After 3 days, the effect on cell viability and the CSC population was analyzed. A significant reduction of the ALDEFLUOR⁺ population and cell viability was observed after treatment with repertaxin or anti-CXCR1 antibody, but not with anti-CXCR2 antibody. (D) After 4 days of treatment, the number of apoptotic cells was evaluated, and 36% of apoptotic cells (green) were detected in repertaxin-treated cells compared with controls, in which mostly viable cells (blue) were present. Scale bars: 100 μm. (E) To determine whether cell death was mediated via a bystander effect, CXCR1⁺ and CXCR1^– populations were treated with various concentrations of repertaxin. A decrease in cell viability in CXCR1⁺ and unsorted populations were detected, whereas no effect was observed in the CXCR1^– population. (F) Serial dilutions of dialyzed conditioned medium from CXCR1⁺ cells treated for 3 days with repertaxin was used to treat sorted CXCR1⁺, CXCR1^–, or unsorted populations. After 2 days of treatment, a massive decrease in cell viability was observed in both CXCR1^– and unseparated populations, whereas no effect was observed in the CXCR1⁺ population. Error bars represent mean ± SD.