lysates were boiled in SDS-PAGE sample buffer and immunoblotted with polyclonal anti-Bax 62M (Metcalfe et al., 1999), anti-calnexin (Stress Gen), and anti-active caspase 3 (a kind gift from Anu Srinvasan, Idun Pharmaceuticals, Inc., La Jolla, CA), and monoclonal rat anti-Bax (Phar- Mingen), mouse anti–Bcl-x (Transduction laboratories), and mouse anti- COX I (Molecular Probes). Microinjection GST fusion proteins were expressed in E. coli and purified on glutathione- agarose (Sigma) as previously described (Gilmore and Romer, 1996). FSK-7 cells were grown to confluence on coverslips before microinjecting with ei- ther GST alone or with the GST-tagged dominant negative pp125FAK Figure 1. Cultured primary mammary epithelial cells and mam- mary cell lines undergo apoptosis after detachment from ECM. (a) Confluent primary mouse mammary epithelial cells and FSK-7 cells were detached by trypsinization and maintained on poly- HEMA coated dishes for the indicated times before low molecu- lar mass DNA was extracted and analyzed on 1.2% agarose gels stained with ethidium bromide. (b and c) Cells were incubated in the absence of adhesion on poly-HEMA coated dishes for the in- dicated times before being cytospun onto glass slides. The num- ber of apoptotic cells was determined by Hoechst 33258 staining and counting of cells with typically apoptotic nuclear morphol- ogy, indicated by the arrows in c. Error bars represent SEM. (DN-FAK) fusion protein at 3 mg/ml in 75 mM KCl, and 10 mM potas- sium phosphate, pH 7.5. Cells were fixed in 2% paraformaldehyde either 1 or 5 h postinjection before immunostaining. Transient Transfections The plasmid pSG5.p110CAAX was a generous gift of Dr. Julian Down- ward (ICRF, London, UK). TS-pp60src was kindly given by Dr. Ged Brady (University of Manchester, Manchester, UK). Both were subcloned into the expression vector pCDNA.3 to produce pCDNA.3/p110CAAX and pCDNA.3/src. pCMV3RDp85 (referred to in text as p85DSH2) was kindly provided by Dr. Phill Hawkins (Babraham Institute, Cambridge, UK). Full-length murine Bax was cloned by PCR using pfu DNA poly- merase (Stratagene) from RNA isolated from adult mouse mammary gland using PCR primers directed against the 59 and 39 ends of the coding sequence. HA-tagged Bax and Bax truncated at its carboxyl terminus at residue 172 (BaxDCT) were generated by PCR using the 59 primer ATG- TACCCATACGACGTCCCAGACTACGCCATGGACGGGTCC, in- corporating the HA epitope tag. TCAGCCCATCTTCTTCCAGAT was used as the 39 primer for Bax, and TCACTGCCATGTGGGGGTCCC for BaxDCT. Both were cloned into pCR-script SK1 (Stratagene) and con- firmed by double stranded sequencing, before subcloning into pCDNA.3 to produce pCDNA.3/HA-Bax and pCDNA.3/HA-BaxDCT. GST-tagged DN-FAK (amino acids 839-1052) was amplified by PCR using the 59 primer GCCGCCATGTCCCCTATACTA, and the 39 primer TCAGTGTGGC- CGTGTCTG, and cloned into pCDNA.3. FSK-7 cells plated onto coverslips at 1 3 105 cells/cm2 were grown to 80–90% confluence before transfecting using lipofectamine plus (GIBCO BRL). Cells were transfected with a total of 3 mg DNA. For cotransfec- tions, 2 mg of pCDNA.3/DN-FAK was used with 1 mg of pCDNA.3, pCDNA.3/p110CAAX or pCDNA.3/src. Cells were transfected for 3 h followed by 18 h incubation in growth medium. Detached cells were col- lected and cytospun onto polysine-coated slides. Both the adherent and the detached cells were immunostained. DN-FAK– or p85DSH2-express- ing cells with apoptotic morphology were counted. Immunofluoresence Cells were fixed in 2% paraformaldehyde in PBS and permeabilized in 0.5% Triton X-100. Cells were stained with anti-Bax 62M, anti-GST (Phar- macia) or the p85 subunit of PI 3-kinase (Upstate Biotechnology Inc.) in PBS with 0.1% horse serum, followed by either Cy2- or Cy3-conjugated secondary antibodies (Jackson Laboratories). Cells were counterstained with 4 mg/ml Hoescht 33258. Cells were viewed on a Zeiss Axiophot photo- microscope equipped with epifluorescence and images were taken on T-MAX 400 film. For comparison of Bax staining, all exposures and sub- sequent image manipulations were identical. For visualization of mito- chondria, cells were incubated for 15 min before fixation with 500 nM Mi- totracker green-fm (Molecular Probes). Results Detachment-induced Apoptosis in Mammary Epithelial Cells Is Preceded by Redistribution of Bax from a Cytosolic to an Insoluble Fraction Mammary epithelial cells require integrin-mediated adhe- sion to ECM for survival (Streuli and Gilmore, 1999). Pri- mary mammary epithelial cells have been shown to un- dergo apoptosis when plated onto an inappropriate ECM (Pullan et al., 1996; Farrelly et al., 1999). This dependence on ECM was confirmed by the rapid onset of apoptosis when mammary cells were detached from their substrata and maintained in suspension by plating onto nonadhesive poly-HEMA. After detachment, nucleosomal DNA ladders were detectable after 3–5 h, along with a loss of cell num- ber and increase in the proportion of cells showing mor- phological changes associated with apoptosis (Fig. 1). De- tachment therefore served to synchronize apoptosis in ECM-dependent mammary epithelial cells. We examined a number of mammary cell lines and found that these also Gilmore et al. Integrin Regulates Apoptosis through Bax Activation 433