showed a strong dependence upon ECM for survival. The mouse mammary cell line, FSK-7, showed rapid apoptotic laddering when maintained on poly-HEMA (Fig. 1 a), with a time course similar to that observed for primary cells. This occurred with a loss of 60% in cell number by 24 h, and an increase in the number of cells showing the con- densed and fragmented nuclei indicative of apoptosis (Fig. 1, b and c). We wished to investigate the possible role of members of the Bcl-2 family in the ECM regulation of mammary cell survival. FSK-7 cells express a number of the known Bcl-2 family proteins, including Bax and Bcl-x. The major pro-apoptotic member of this family in FSK-7 cells, Bax, has previously been shown to display a cytosolic distribu- tion in a number of cell types, but redistributes to mito- chondria when cells are treated with apoptosis-inducing drugs (Hsu et al., 1997). To determine if redistribution of endogenous Bax was involved in apoptosis of mammary cells detached from ECM, we compared Bax localization in FSK-7 cells in monolayer culture and after culture on poly-HEMA. Cells were lysed by swelling in a hypotonic buffer, followed by Dounce homogenization. The efficacy of homogenization was checked by trypan blue staining, and found to be .95%. The soluble and insoluble fractions were separated by centrifugation and the relative distribution of Bax was analyzed by immunoblotting (Fig. 2 a). In healthy mono- layer culture, where apoptotic cells represented ,4% of the population, Bax was predominantly found in the cyto- sol. However, in cells maintained on poly-HEMA for 1 h, Bax showed a marked redistribution to the insoluble frac- tion. The mitochondrial marker cytochrome oxidase I in- dicated that equivalent amounts of the insoluble fractions were loaded, also confirmed using the ER marker protein calnexin. The lack of these proteins in the cytosol indicated that the presence of Bax in the cytosolic compartment was not due to organelle disruption. We also examined the ma- jor pro-survival member of the Bcl-2 family expressed in FSK-7 cells, Bcl-x. This was primarily associated with the in- soluble fraction, both in adherent cells and those in suspen- sion. Thus, loss of adhesion to ECM results in a rapid relo- calization in the subcellular distribution of Bax. Translocation of Bax to internal cell membranes, medi- ated by its carboxy-terminal domain, has previously been shown to be required for Bax apoptotic function in COS-7 cells (Wolter et al., 1997). We confirmed that this was also the case in mammary epithelia by transfecting FSK-7 cells with HA-tagged Bax with or without the hydrophobic car- boxyl terminus (HA-Bax and HA-BaxDCT). Transfected cells were examined for apoptotic morphology (Fig. 2 b). We found that overexpression of HA-Bax induced three times more apoptosis than HA-BaxDCT. These results in- dicate that in FSK-7 cells, membrane targeting of Bax is required for it to induce apoptosis. The redistribution of Bax occurred within 1 h after detach- ment from ECM, well before the appearance of apoptotic markers such as nuclear condensation. Bax remained associ- ated with the insoluble fraction of cells throughout pro- longed culture in suspension, while cells were undergoing apoptosis (Fig. 2 c). Redistribution of Bax was not associated with any change in the total amount of the protein detected, and was, therefore, unlikely to require the de novo synthesis of Bax. This was confirmed by treating cells for 1 h before detachment from ECM with cycloheximide, which had no effect on its redistribution to the insoluble fraction (Fig. 2 d). Bax Redistribution Occurs to Mitochondria To determine whether Bax translocated to an identifiable subcellular compartment, cells were stained with a poly- clonal antibody raised against a peptide corresponding to amino acids 44–59 of Bax (62M) (Metcalfe et al., 1999). Figure 2. Subcellular distribution of Bax in mammary epithelial cells is altered after de- tachment from ECM. (a) Adherent FSK-7 cells were either maintained on tissue culture dishes or were detached from ECM and incu- bated on poly-HEMA for 1 h. Cells were frac- tionated into insoluble (Insol) and cytosolic (Cyt) fractions as described in Materials and Methods. Fractions were analyzed by immu- noblotting for Bax, Bcl-x, calnexin, and cyto- chrome oxidase subunit I (COX I). Similar re- sults were obtained for primary mammary epithelial cells. Data are representative of mul- tiple experiments. (b) FSK-7 cells were trans- fected with 2 mg of pCDNA.3, pCDNA.3/ HA-Bax, or pCDNA.3/HA-BaxDCT. 18 h af- ter transfection cells were stained with Hoechst 33258, anti-HA mAb and 62M anti-Bax. Transfected cells were scored for percent apoptotic cells based on nuclear morphol- ogy. (c) FSK-7 cells were detached from ECM and maintained on poly-HEMA for 5 and 12 h before homogenization and separation into insoluble and cytosolic fractions. Fractions were analyzed by immunoblotting with 62M anti-Bax and monoclonal anti–Bcl-x. (d) FSK-7 cells were treated with 25 mg/ml cyclohex- imide for 1 h before detachment from ECM for 3 h. Cells were fractionated and Bax localization analyzed by immunoblotting with 62M. The Journal of Cell Biology, Volume 149, 2000 434