Fig. 5. MG132 protected against mitochondrial permeabilization in cells.

293T cells expressing GFP/BimS-ER were pre-incubated for 1 h with 100 µM of zVAD-(Ome)-fmk and/or 10 min with 100 µM of MG132, prior to the addition of 1 µM of 4-OHT. After an incubation of 8 h at 37° C, cells were fixed and stained for cytochrome c. a. MG132 inhibited cytochrome c release. Confocal images were acquired using Lasersharp 2000 software. Arrowheads highlight cells in which cytochrome c has been released from mitochondria. b. MG132 did not inhibit loss of Opa1 complexes. Mitochondria from the samples treated as above were cross-linked with EDC and Western blotted with antibodies against OPA1, Hax 1 and cytochrome c. For blots with Mcl1, total cell lysates were used. c. MG132 inhibited cytochrome c release at high concentrations, but not at lower concentrations that still inhibit proteasomal protein degradation. The percentage of cells with released (diffuse) cytochrome c was determined by cell counts. The experiments were performed in triplicate. Inhibitors used are indicated at the bottom of the graph.