Fig. 2

Caspase inhibition only partially attenuated cytokine-induced cytotoxicity, while it almost completely blocked Fas ligand-induced cytotoxicity. A549 cells were pretreated with DMSO (vehicle) or QVD-OPh (pan-caspase inhibitor) 1 h before CM or Fas ligand (Fas L; specific apoptosis inducer) stimulation. Forty-eight hours after each stimulation, cytotoxicity and apoptosis were evaluated as described in the “Methods” section. a Grey bar graph shows cytotoxicity evaluated by monitoring the concentration of released LDH in culture medium as described in the “Methods” section. Black bar graph shows caspase activation estimated by determining the concentration of cleaved cytokeratin 18 in culture medium using an M30 cytodeath ELISA kit as described in the “Methods” section. QVD-OPh only partially blocked CM-induced cytotoxicity (determined by LDH), although it almost completely blocked the apoptotic component (termed as “apoptotic index”) estimated by cleaved cytokeratin18. On the other hand, LDH increase by specific apoptotic pathway stimulation (using Fas ligand; Fas L) was almost completely inhibited by QVD-OPh as well as cleavage of cytokeratin18. Bar graph shows means ± SEM. *P < 0.05 vs. CM alone, **P < 0.01 vs. CM alone, ^#P < 0.01 vs. Fas L alone. b Total cell lysates were collected and then subjected to western blotting. Caspase 7 cleavage and phosphorylation of histone H2AX were significantly blocked by QVD-OPh pretreatment in CM stimulation as well as Fas ligand stimulation