Figure 3. Detachment- induced Bax relocalization occurs to mitochondria and coincides with a conforma- tional change that exposes its BH3 domain. (a) Adherent FSK-7 cells and detached cells maintained on poly- HEMA for the indicated times before cytospinning onto polysine slides, were immunostained with anti- Bax 62M and Hoechst 33258. (b) FSK-7 cells on poly- HEMA for 1 h were in- cubated with Mitotracker before cytospinning and immunostaining for Bax. (c) Adherent cells or cells on poly-HEMA for the indi- cated times were treated for 30 min with either the cell permeable cross-linker DSS or carrier alone. Total cell ly- sates were then analyzed by immunoblotting for Bax or Bcl-x. No change in Bcl-x immunoreactivity was ob- served after DSS treatment. Results are representative of three experiments. This region of Bax is at its amino terminus and close to the BH3 domain critical for its pro-apoptotic function (Hunter and Parslow, 1996). FSK-7 cells maintained in suspension for 1, 6, and 24 h were cytospun onto polysine-coated slides and Bax staining was compared with that in an ad- herent monolayer of cells (Fig. 3 a). Detachment from ECM resulted in a marked increase in Bax staining, which showed a punctate distribution within the cytoplasm. This occurred within 1 h of detachment, on a time course iden- tical with the observed translocation of Bax to the insolu- ble fraction of the cells, and preceding the onset of apopto- sis, seen through the condensation and fragmentation of nuclei at 6 and 24 h (compare Fig. 3 a with 1 b). To deter- mine if Bax localized to mitochondria, detached cells were Gilmore et al. Integrin Regulates Apoptosis through Bax Activation 435