Figure 4

VDAC1 silencing blocks TRAIL induced mitochondrial apoptosis. (A) Western blot of BAX in BMH-crosslinked mitochondrial fraction from TRAIL-treated or time-matched cells. Dimers and trimers of BAX indicating BAX activation appear in NT cells following TRAIL but this is reduced in VDAC1 knockdown cells. (B) Western blot of BID in sh-NT and sh-VDAC1 cells. Full length p23-BID is activated by caspase-8 mediated cleavage in mitochondria following TRAIL treatment in sh-NT control cells but is not processed in the VDAC1 knockdown cells. We were unable to detect the 16 kDa tBID fragment by western blot. COX4 is shown as loading control. (C) Western blot of cytochrome-C in mitochondrial fraction of sh-NT and sh-VDAC1-2A cells. Cyt-C is lost from sh-NT control mitochondria following TRAIL, indicating mitochondrial outer membrane permeabilization (MOMP). MOMP is not observed in VDAC1 knockdown cells. (D) Mitochondria were extracted from cells shown and were resuspended in respiration buffer and stained with JC-1 dye (see methods) to measure mitochondrial polarization status. 25 μg of mitochondria were aliquoted per well of 96-well plate (n = 5) and incubated with recombinant caspase-8-cleaved BID (tBID) at concentrations shown for 45 minutes. 1% Triton-X treatment was used as positive control for full depolarization. Ratio of green (530 nm) to red (580 nm) fluorescence of JC-1 increases as mitochondria depolarize.