Figure 5

VDAC1 is required for FAS ligand-induced and FLIP silencing-induced activation of caspase-8. (A) Clonogenic assay shows impairment of cell regrowth in sh-NT and sh-VDAC1 cells following exposure to TRAIL or CH11. Clonogenicity is significantly reduced in sh-VDAC1 cells compared to sh-NT cells for both death ligands. (B) Cells were treated with 150 ng/ml FAS ligand CH11. Western blot shows that caspase-8-p18 is generated in sh-NT cells following treatment with CH11, but this is significantly reduced in sh-VDAC1 cells. (C) sh-NT or sh-VDAC1 cells were transfected with c-FLIP, or scrambled control siRNA. Caspase-8-p18 was detectable 24 h post FLIP siRNA treatment in sh-NT cells, but not sh-VDAC1 cells. H460 cells were generated that stably expressed the coding region only of VDAC1 fused to myc epitope tag (pVDAC1-5A or pVDAC1-5B), or empty vector (EV). (D) DEVDase assay showing increased caspase-3 activity in pVDAC1 cells following 10 ng/ml TRAIL treatment, which is unaffected by V1-3' siRNA, whereas control cells show reduced caspase-3 activity in comparison, which is significantly attenuated by V1-3'siRNA. (E) Western blot shows expression of Myc tag and a moderate increase in overall VDAC1 in pVDAC1-5A and pVDAC1-5B. (F) siRNA targeting 3'UTR of VDAC1 [V1(3')] causes moderate VDAC1 knockdown in EV cells, but not in pVDAC1 cells. TRAIL treatment shows that V1(3') siRNA reduces processing to Casp-8 (p18) in EV cells. (G) Myc-tagged VDAC1 expressing cells exhibit increased sensitivity to TRAIL as shown by increased processing of PARP.