Lysates prepared as described above were separated by SDS PAGE under reducing conditions followed by trans fer to a 0. 45 um PVDF membrane. Non specific binding was blocked by one hour incubation at room tempera ture in TBS T con taining 5% of blocking reagent. Primary monoclonal anti bodies were incubated for one hour at 37 C. After 3 washes with TBS T, membranes were incubated with pero idase conjugated secondary antibody for one hour at 37 C. Following 3 washes with TBS T, blots were revealed using the chemiluminescent blotting Substrate Kit. Cell death assays Following the indicated treatments, cells were labeled with the IOTest anti APO2. 7 PE according to the manufacturers instructions. Briefly, floating and adherent cells were washed once in PBS, transferred in 96 well plates and washed twice more in cold PBS.

Cells were then resuspended in 500 ul of labeling mi diluted in PBS and incubated in the dark for 15 minutes at RT. Cells were then washed in PBS and either immediately analyzed by FACS or fi ed in 1% paraformaldehide for delayed FACS analysis. APO2. 7 positive cells were analyzed using the FL1 channel of a FACS CaliburTM cytofluorometer. Anne in V staining was performed similarly, according to the manufac turers instructions. Mammosphere assays BT474 cells treated with the indicated siRNA were plated as single cells in ultra low attachment plates at low density. They were grown in serum free mammary epithelial cell growth medium containing DMEM F12 supple mented with B27 and MEGM singlequots, as previously described.

Mammo sphere forming unit were counted as number of mam mospheres 50 mm. Chromatin Immunoprecipitation assays BT474 cells treated or not with RAD001 Drug_discovery were washed and cross linked with formaldehyde at room temperature for 8 min essentially as previously described. Reaction was stopped with 10 ml of 125 mM glycin solution. Cells were washed with cold PBS and lysed in 500 ul of lysis buffer, and sonicated five times for 20 seconds each. Supernatants were then recovered by centrifugation at 12 000 rpm for 10 min at 4 C, diluted once in dilution buffer and subjected to one round of immunoclearing for 2 h at 4 C with 2 ug of sheared sal mon sperm DNA, and 20 ul of proteinG agarose coated with salmon sperm DNA. Immunoprecipitation was performed overnight with specific antibodies and IgG control, and then 2 ug of sheared salmon sperm DNA and 20 ul of proteinG agar ose coated with salmon sperm DNA were further added for 1 h at 4 C.

Note that immunoprecipitations were performed in the presence of 1% Igepal CA 630. Immunoprecipitates were washed sequentially for 10 min each in TSE I, TSE II, and TSE III. Beads precipi tates were then washed once with TE buffer and eluted once with 1% SDS, 100 mM NaHCO3. Eluates were heated at 65 C for 6 hours to reverse the formaldehyde cross linking. DNA was precipitated using classical pro cedures.