Significant but lower increases in PC PLC content and activity were HTS also found in other BC cell lines. The rates of PC PLC and SMS activity were measured in MDA MB 231 cells in either the presence or absence of D609. Special traits of MET and BC cell differentiation such as decreased expression Inhibitors,Modulators,Libraries of vimen tin and N cadherin, downmodulation of molecules criti cally involved in tumor progression, such as galectin 3 and milk fat globule epidermal growth factor 8, and production of b casein were detected in D609 treated MDA MB 231 cells, together with long standing and irreversible reduction of in vitro cell moti lity and invasion capabilities. Typical features of cell dif ferentiation, such as proliferative arrest with maintenance of cell viability, changes in cell morphol ogy, and formation of lipid bodies, were induced by D609 in all of the investigated BC cells.

Materials and methods Cells The human BC cell lines MDA MB 231, SKBr3, and MCF 7 and the non tumorigenic immortalized human mammary epithelial cell line MCF 10A were supplied by American Type Culture Collection. The cells were cultured, as previously described, in either the pre sence or Inhibitors,Modulators,Libraries absence of D609. Antibodies and reagents Rabbit polyclonal antibodies raised against bacter ial PC PLC and selectively cross reacting with mammalian PC PLC were obtained in our laboratory in accordance with a modification of the method originally described by Clark and colleagues and characterized as reported.

Alexa Fluor 633 conjugated phalloidin, 4,4 difluoro 1,3,5,7,8 pentamethyl 4 bora 3a, 4a diaza s indacene, Bodipy TR ceramide, and the secondary Inhibitors,Modulators,Libraries Abs Alexa Fluor 594 Inhibitors,Modulators,Libraries F 2 fragments of goat anti rab bit and goat anti mouse IgG were purchased from Molecular Probes Inc. mouse anti b actin and anti vimentin Abs from Sigma Aldrich, rabbit poly clonal anti HER2, anti E cadherin, and anti N cadherin and mouse monoclonal anti MFG E8 from Santa Cruz Biotechnology, Inc. monoclonal anti galectin 3 and anti b casein Abs from Abcam, and horseradish peroxidase conju gated goat anti mouse and goat anti rabbit IgG from Bio Rad Laboratories, Inc. Chemi cals were from Sigma Aldrich unless otherwise specified. Confocal laser scanning microscopy and flow cytometry analyses For immunofluorescence analyses, cells were seeded in 24 well cluster plates onto 12 mm cover glasses.

After Inhibitors,Modulators,Libraries 48 hours of culture in complete medium, cells were treated with or without D609 for different times, fixed in 3% paraformaldehyde, permeabi lized by Triton X 100, and then stained at 37 C with Bodipy 493 503, followed by Alexa Fluor 633 conjugated phalloidin or by the primary and Alexa Fluor 594 conjugated sec ondary definitely Abs. The cover glasses were finally mounted on the microscope slide with Vectashield anti fade mount ing medium containing 4 6 diamidino 2 phenylindole.