Requirement of Fas/CD95 for ALP-induced apoptosis. (A) Cell-surface expression of Fas/CD95, DR4, and DR5 in MM144, OPM-2, and Fas/CD95-deficient OPM-2 cells was determined by flow cytometry. Percentage of positive cells for each death receptor was estimated using the P3X63 myeloma culture supernatant and an isotype-matched FITC-conjugated nonrelevant IgG monoclonal antibody as negative controls. (B) Edelfosine uptake was determined after incubating MM144, OPM-2, and Fas/CD95-deficient OPM-2 cells with 10 nmol [³H]edelfosine for 6 hours. (C,D) Fas/CD95-deficient OPM-2 and MM144 cells were untreated (control) or treated with 10 μM edelfosine or perifosine for 12 hours, and then Fas/CD95 cell-surface expression was determined by flow cytometry as the percentage of Fas-positive cells (C) or MFI (D). (E) Cell-surface expression of Fas/CD95 was analyzed by immunofluorescence flow cytometry in Fas/CD95-deficient OPM-2 infected with empty virus (Fas-deficient OPM-2–vector), recombinant retrovirus containing human Fas/CD95 (Fas-deficient OPM-2–Fas), and recombinant retrovirus containing a truncated version of human Fas/CD95 (Fas-deficient OPM-2-FasΔ57C), lacking the 57 COOH-terminal amino acids. Percentage of Fas/CD95⁺ cells was estimated using the P3X63 myeloma supernatant and an isotype-matched FITC-conjugated nonrelevant IgG monoclonal antibody as negative controls. (F) Induction of apoptosis in the above retrovirus-infected cells was determined by flow cytometry after a 24-hour incubation with 10 μM edelfosine or perifosine. Untreated control cells were run in parallel. Data shown are means ± SD of 3 independent experiments.