(A) To determine whether the bystander killing effect induced by repertaxin treatment was mediated by FASL, we measured the level of soluble FASL in the medium using an ELISA assay. After 4 days of treatment, a greater than 4-fold increase of soluble FASL was detected in the medium of cells treated with repertaxin compared with untreated controls. (B) We measured the level of FASL mRNA by RT-PCR and confirmed the increase of FASL production after treatment with repertaxin. Similar results were observed after 4 days of treatment with a FAS agonist that activates FAS signaling, with a 5-fold increase of the FASL mRNA compared with control. (C) SUM159 cells were cultured in adherent conditions and treated with repertaxin alone or in combination with anti-FASL. Interestingly, cell growth inhibition induced by repertaxin treatment was partially rescued by addition of anti-FASL. Moreover, cells treated with a FAS agonist displayed cell growth inhibition similar to that of cells treated with repertaxin alone. (D and E) The effect of repertaxin treatment, alone or in combination with anti-FASL, and FAS agonist treatment on the CXCR1⁺ and ALDEFLUOR⁺ population was analyzed. The massive decrease in the CXCR1⁺ and ALDEFLUOR⁺ population induced by repertaxin treatment was not rescued by the anti-FASL, and treatment with FAS agonist produced 10- and 3-fold increases in the percent of the CXCR1⁺ and ALDEFLUOR⁺ populations, respectively. BAAA, BODIPY aminoacetaldehyde; DEAB, diethylaminobenzaldehyde. Error bars represent mean ± SD.