incubated with the mitochondrial stain Mitotracker green- fm before fixation and immunostaining with the 62M anti- body. This is incorporated into the mitochondria of live cells, but is retained after aldehyde fixation. Bax immuno- fluorescence in detached cells was found to coincide with Mitotracker staining (Fig. 3 b). The relocation of Bax to an insoluble fraction after detachment from ECM therefore coincided with the exposure of its BH3 domain and its ap- pearance within mitochondria. To verify that the conformation of the Bax BH3 epitope altered during detachment-induced apoptosis, living FSK-7 cells were treated with the membrane permeable cross- linker DSS. Cells were treated with DSS before lysis, and compared with untreated cells. In the absence of cross- linking, there was no change in the total amount of Bax present over the time course of the experiment. However, we observed very little Bax immunoreactivity with the 62M antibody in whole cell lysates after cross-linking of proteins within monolayers of cells adherent to ECM (Fig. 3 c). After detachment, however, Bax immunoreactivity in- creased with a time course similar to that observed both for redistribution of Bax to the insoluble fraction and for the in- crease in Bax immunofluorescence. A similar result was seen with a second anti-Bax antibody (data not shown). As with Bax translocation, the increase in Bax immunoreac- tivity after cross-linking was not blocked by treating cells on Figure 4. Detachment- induced Bax redistribution precedes activation of caspases and cytochrome c release. (a) 100 mM of the caspase in- hibitors, DEVD or zVAD-fmk, were added 1 h before de- tachment of FSK-7 cells from ECM, and throughout the in- cubation on poly-HEMA for 3 h. Cells were homogenized in hypotonic buffer and in- soluble and cytosolic frac- tions were analyzed with the 62M anti-Bax antibody. (b) FSK-7 cells with or with- out caspase inhibitors were maintained on poly-HEMA for 1 h before immunostain- ing for Bax. (c) FSK-7 cells were maintained on poly- HEMA for 24 h in the pres- ence or absence of either zVAD or DEVD. Cells were cytospun onto glass slides be- fore immunostaining with anti-activated caspase 3 and Hoechst 33258. Both the per- centage of apoptotic cells and the number of cell re- maining were determined. Caspase inhibitors reduced both the loss of cell number and the percentage apoptosis in the remaining cells. (d) FSK-7 cells were left at- tached to ECM or detached and maintained in the pres- ence or absence of DEVD. Whole cell lysates were analyzed by immunoblot- ting with the antibody to activated caspase 3, CM1. (e) FSK-7 cells, either adher- ent or detached and main- tained on poly-HEMA for 3 h, were fractionated and the dis- tribution of cytochrome c com- pared with Bax. The Journal of Cell Biology, Volume 149, 2000 436