TAK1 inhibition triggers TRAIL-induced death in OCs and potentiates it in MM cells. (A) Mature OCs from RAW264.7 cells and mouse BMMs were cultured in the presence or absence of LLZ1640-2 (LLZ) at 300 nM for 48 hours. TRAIL was added at the indicated concentrations. (Ai) The numbers of TRAP-positive MNCs were counted. (Aii) Percent distribution of TUNEL-positive cells was also estimated within OCs derived from mouse BMMs and human PBMCs. Data were expressed as mean ± SD. *P < .05. (Aiii) The protein levels of c-FLIP, caspase-8, and cleaved caspase-8 were analyzed by western blotting. β-actin was used as a protein loading control. (B) Mature OCs from RAW264.7 cells were cultured in the presence or absence of LLZ at 150 nM for 48 hours. TRAIL at 100 ng/mL and the caspase-8 inhibitor Z-IETD-FMK at 100 μM were added as indicated. The protein levels of caspase-8, cleaved caspase-8, Sp1, and c-FLIP were analyzed by western blotting. β-actin was used as a protein loading control. (C) MM cell lines, RPMI 8226, MM.1S, and 5TGM1, were cultured in the presence or absence of LLZ at 300 nM for 48 hours. TRAIL was added at the indicated concentrations. The cell viability was measured by WST-8 cell proliferation assay. Results were expressed as the mean ± SD. *P < .05. (D) The indicated MM cell lines were cultured in the presence or absence of LLZ at 300 nM for 6 and 24 hours to perform RT-PCR and western blotting, respectively. TRAIL was added at 100 ng/mL as indicated. The protein levels of caspase-8, cleaved caspase-8, Sp1, and c-FLIP were analyzed by western blotting (left). β-actin was used as a protein loading control. Z-IETD-FMK was added at 100 μM as indicated. c-FLIP mRNA expression was analyzed by RT-PCR. GAPDH served as an internal control.