The low invasive and highly invasive breast cancer cells were purchased from American Type Culture Collection. These cells were cul tured in Dulbeccos modified Eagles medium and leibovitzs L 15 supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 ug/ml streptomycin and 2 mM glutamine in a Fluoro-Sorafenib humidified atmosphere at 37 C. Plasmids and DNA Transfection The wild type and rapamycin resistant mTOR in pIRES GFP expression vector were a generous gift from Dr. Rok Humar. The wild type and rapamycin resistant HA S6K1 in pRK7 expression vector were kind gift from Dr. John Blenis. The super repressor form of I��B fused downstream to a FLAG epitope in an expression vector was Inhibitors,Modulators,Libraries a gift from Dr. Dean Ballard. The wild type pCEFL GFP c Fos was a kind gift from Dr. Omar A Coso.

The dominant negative c Fos in pCMV500 expression vector Inhibitors,Modulators,Libraries was a kind gift from Dr. Nicole Darack. The wild type c Jun in pRJB10B expression vector and dominant negative c Jun in pELFIN expression vector were kind gifts from Dr. Jalam. The ICAM 1 Luc construct was a kind gift from Dr. Arshad Rahman. The MCF 7 cells were transiently transfected with cDNA using Lipofectamine Inhibitors,Modulators,Libraries 2000 according to man ufacturers instructions. Transfected cells were used for ICAM 1 expression, NF ��B and AP 1 DNA binding, NF ��B, AP 1 and ICAM 1 luciferase assays and p70S6 kinase phosphorylation studies. Western Blot Analysis For ICAM 1 expression, MCF 7 and MDA MB 468 cells were treated with OPN in a time and dose dependent manner. In separate experiments, MCF 7 cells were either transfected with various cDNA constructs or pre treated with 20 nM rapamycin for 1 h and then treated with 0.

5 uM OPN and level of ICAM 1 was detected. For p70S6 kinase and mTOR phosphorylations, cells were treated with 0. 5 uM OPN for 0 120 min. In other experi ments, the cells were either transfected with mTOR Inhibitors,Modulators,Libraries con structs or pretreated with 20 nM rapamycin or 0 500 uM U0126 for 1 h and then treated Inhibitors,Modulators,Libraries with 0. 5 uM OPN. The cells were lysed in lysis buffer, 150 mM NaCl, 1% Nonidet P 40, 0. 5% sodium deoxy cholate, 5 mM dithiothreitol and 1 mM phenylmethylsul fonyl fluoride) and the protein concentrations in cleared supernatants were measured by using Bio Rad protein assay. The supernatant containing equal amount of total proteins were resolved by SDS PAGE and electrotransferred from gel to nitrocellulose membranes.

The membranes were incubated with anti p p70S6K, anti p mTOR, anti p ERK1/2 or anti ICAM 1 antibodies and further incubated with horseradish peroxidase con jugated IgG and detected by luminol reagent according to the mean manufacturers instruction. The same blots were re probed with anti actin or non phospho antibodies of respective molecules and detected. Nuclear Extracts and Electrophoretic Mobility Shift Assay The NF ��B and AP 1 EMSA were performed as described earlier. Briefly, MCF 7 cells were treated with 0. 5 uM OPN for 0 240 min at 37 C.