VDAC1 silencing blocks TRAIL induced apoptosis. (A) Western blot showing H460 clones with stable knockdown of VDAC1 by different shRNA sequences (V1-1B and V1-2A), and control cells expressing non-targeting shRNA (NT). (B) ATPase viability assay showing reduction in viability 24 h following 10 ng/ml TRAIL treatment, which is significantly attenuated in VDAC1 knockdown clones compared to NT control cells (*p < 0.0001 v sh-NT). (C) Flow cytometry of PI-stained H460 clones following TRAIL treatment. Apoptotic cells are indicated by increase in sub-G0/G1 population (*p < 0.05). (D) Luminescent DEVD-ase assay measures caspase-3-like activity in sh-NT and sh-VDAC1 cells 24 h after TRAIL treatment (10 ng/ml), shown relative to time-match control for each cell line. Increase in caspase-3 activity in NT control is significantly attenuated in sh-VDAC1 cells (*p < 0.0005 v sh-NT). (E) Western blot for PARP in sh-NT and sh-VDAC1 cells following TRAIL treatment. PARP cleavage occurs in sh-NT control cells but not sh-VDAC1-1B or sh-VDAC1-2A cells. (F) Western blot of H460 cells transiently transfected with siRNA to VDAC1 (V1) or Non-Targeting siRNA (NT). (G) Transient siRNA knockdown of VDAC1 was carried out and cells were treated with TRAIL. DEVD-ase assay showing significant attenuation of caspase-3-like activity following VDAC1 knockdown.