TRAIL induces cell death in MM cells but not in OCs. (A) Human MM cell lines, MM.1S, INA-6, and TSPC-1, and murine 5TGM1 MM cell line were treated in triplicate with the indicated concentrations of TRAIL for 48 hours. Cell viability was determined by WST-8 assay. Data were expressed as mean ± standard deviation (SD). *P < .05. (B) To generate mature OCs, RAW264.7 cells were cultured for 4 days with sRANKL at 50 ng/mL, BMMs for 7 days with a combination of M-CSF (20 ng/mL) and sRANKL (50 ng/mL), and human PBMCs for 14 days in the presence of M-CSF (10 ng/mL) and sRANKL (50 ng/mL). Mature OCs from RAW264.7, mouse BMMs, and human PBMCs were treated in triplicate with the indicated concentrations of TRAIL for 48 hours. Representative results are shown. The numbers of TRAP-positive MNCs were counted (upper, left). Cell viability was also determined by TUNEL assay (upper, right). Data were expressed as mean ± SD. The experiment was performed by 3 independent experiments with triplicate. Mature OCs from RAW264.7 cells were treated with TRAIL (500 ng/mL) for 48 hours, and F-actin and nuclei were stained with phalloidin (green) and 4’,6-diamino-2-phenylindole (blue), respectively (lower, left). Samples were visualized with a fluorescence microscope (BX50, Olympus). Original magnification ×100. Bar represents 100 μm. MNCs with an intact actin ring were counted (lower, right). (C) Mature OCs from RAW264.7 cells and MM.1S were starved with 1% FBS for 24 hours, and then incubated with TRAIL at 100 ng/mL for the indicated periods. For immunoprecipitation, cell lysates were collected and an equal amount of each protein lysate was incubated with anti-DR5 antibody. DISC formation was analyzed by western blotting with anti-FADD, anti-caspase-8, and anti-DR5 antibodies. (D) Mature OCs from RAW264.7 cells and the indicated MM cell lines were treated with TRAIL at 100 ng/mL for 48 hours. The protein levels of c-FLIP were analyzed by western blotting. β-actin was used as a protein loading control.