VDAC1 silencing blocks cleavage of caspase-8 to its p18 subunit. (A) Confocal microscopy image showing colocalisation of Caspase-8 (green) with the mitochondrial marker protein COX-4 (red) in H460 cells. Colocalisation is indicated by yellow staining in merged image. (B) Caspase-8 western blot of mitochondrial and cytosolic fractions of cells transiently transfected with NT siRNA or VDAC1 siRNA. Catalytically active caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following TRAIL. Caspase-8-p18 is predominantly in mitochondrial fraction, but excluded from cyotsolic fraction. COX4 blot is shown as mitochondrial marker; α-tubulin is shown as cytosolic marker. (C) Caspase-8 western blot on mitochondrial fraction from H460 shRNA clones treated with 10 ng/ml TRAIL for 24 h. Appearance of 18 kDa form of caspase-8 is strong in mitochondrial fraction of sh-NT control cells but is abolished in sh-VDAC1-1B and sh-VDAC1-2A cell lines. COX4 is shown as mitochondrial loading control. (D) Caspase-8 western blot of cell lysates from MOR cell line transfected with NT siRNA or VDAC1 siRNA. Caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following 10 ng/ml TRAIL treatment. (E) Caspase-8 western blot of cell lysates from SKMES cell line transfected with NT siRNA or VDAC1 siRNA. Increase in caspase-8 activation to p43 isoform is detected in NT-siRNA cells following 50 ng/ml TRAIL, but not in VDAC1-siRNA cells. Caspase-8 p18 was not detectable in SKMES cell line at reasonable concentrations of TRAIL.