of adherent FSK-7 cells induced apoptosis, seen by the ap- pearance of nucleosomal ladders (Fig. 6 a). It also resulted in translocation of Bax from the soluble to the insoluble fraction (Fig. 6 b), but had no effect on the total amount of expressed Bax or the distribution of Bcl-x. In addition, treatment with herbimycin A for 5 h resulted in an in- crease in punctate staining of the Bax BH3 epitope, similar to that observed in cells maintained on poly-HEMA (Fig. 6 c). All the attached cells showing increased Bax staining had normal nuclei, whereas all those cells that had de- tached after 5 h herbimycin A treatment were apoptotic, with condensed and fragmented nuclei, and all showed in- tense punctate Bax staining. Thus, inhibition of tyrosine kinases in adherent cells induced the exposure of the Bax BH3 domain epitope and its translocation from the cytosol to the insoluble fraction, and this preceded the onset of apoptosis. To confirm that protein tyrosine kinases were involved with the adhesion-dependent regulation of Bax transloca- tion and mammary epithelial cell apoptosis, detached cells maintained on poly-HEMA were treated with the protein tyrosine phosphatase inhibitor pervanadate. Pervanadate completely inhibited detachment-induced apoptosis, even after 24 h in suspension (Fig. 6 d). Treatment with pervan- adate also prevented Bax translocation to the insoluble fraction after detachment (Fig. 6 e). Similar results were also seen with orthovanadate alone, and in control experi- ments hydrogen peroxide alone did not suppress apoptosis Figure 6. Adhesion-dependent subcellular local- ization of Bax and apoptosis are regulated by protein tyrosine kinases. (a) Adherent FSK-7 cells were treated with 1 mM herbimycin A in DMSO or with DMSO alone, or were main- tained on poly-HEMA. Cells were treated for 24 h before harvesting and extracting low molecular mass DNA for analysis of nucleosomal ladders. (b) Adherent FSK-7 cells were treated with 1 mM herbimycin A or carrier alone, or maintained on poly-HEMA, for 3 h. Cells were then lysed in hy- potonic buffer and fractionated by Dounce ho- mogenization. Bax localization was analyzed by immunoblotting. Treatment with herbimycin A resulted in an increase in the amount of Bax as- sociated with the insoluble fraction. (c) Adher- ent FSK-7 cells were treated with 1 mM her- bimycin A for 5 h before fixation in 3.7% formaldehyde and staining with anti-Bax 62M and Hoechst 33258 to visualize nuclei. Cells treated with carrier alone only showed weak staining with 62M. Herbimycin A–treated cells showed an increase in the punctate Bax staining as seen in detached FSK-7 cells (compare with Figs. 3 and 4). Treatment of cells with herbi- mycin A resulted in a number of cells detaching from the monolayer. These cells were cytospun onto polysine slides and stained for Bax. All dis- played condensed and fragmented nuclei, typical of apoptosis, and stained strongly for Bax. (d) FSK-7 cells were detached and maintained on poly-HEMA with or without pervanadate. Cells were harvested at 3, 5, and 24 h. Low mo- lecular mass DNA was isolated and separated on 1.2% agarose to assay nuclear laddering. Pervan- adate completely inhibited the appearance of DNA ladders, even after 24 h culture on poly- HEMA. (e) Cells cultured either as an adherent monolayer, or as detached cells on poly-HEMA with or without pervanadate, were lysed in hypo- tonic buffer and fractionated into insoluble and cytosolic fractions. Fractions were separated by SDS-PAGE and analyzed by immunoblotting with anti-Bax 62M and anti–Bcl-x antibodies. Pervanadate-treated cells still show predomi- nantly soluble Bax. Note the apparent reduction in mobility of Bax after pervanadate treatment, which is not seen with Bcl-x. The Journal of Cell Biology, Volume 149, 2000 438