culture MCF-7 and LCC6 cells were cultured according to the literature 6 . MTT assay Cells were plated in triplicate in 4-well plates at a density of 0,000 cells per well in growth media. After 4 h, cells were switched to SFM for 8–4 h. Cells were then treated with Cilostazol various doses of PQIP for 7 h. Growth was estimated using the 3-4,5-Dimethyulthiazol -yl ,5-diphenyltet- raolium bromide (MTT) assay as described previously. Immunoblotting Serum-starved cells were pretreated with PQIP for 30 min and stimulated with 5 nM IGF-I or 0 nM insulin for 0 min at 37 ° C.

Cells lysate were collected and separated by SDS– gels 5 . Proteins were transferred to nitrocellulose and immunoblotted with the various anti- bodies following manufacturers’ instructions. Immunoprecipitation Pre-cleared total cellular lysates were incubated with Dasatinib IGFR or InsR antibody overnight followed by incubation with protein A agarose for 4 h at 4 ° C. Samples were run on SDS– gel, transferred to nitrocellulose, and immu- noblotted for phosphotyrosine residues. Breast Cancer Res Treat Anchorage-independent growth assay Anchorage-independent growth assays were performed as previous described 5 . The bottom agar was overlaid with 800 l l of a 0.45% top agar mixture containing 0,000 LCC6 cells per well in the presence of DOX, PQIP or both, and incubated at 37 ° C for 4 h. The second treatment was given on the top of agar. After 9–0 days, colonies were counted using a light microscope with an ocular grid.

Five random fields were counted per well, and only colonies exceeding two-thirds of a grid square were scored. Cell cycle analysis Confluent MCF-7 cells were plated at a density of 0.4 9 0 6 cells per 60 mm dish. After 4 h, cells were switched to SFM for 4 h. Cells were naratriptan Serotonin receptor inhibitor then treated with PQIP and with or without IGF stimulation. Cells were collected in PBS and stained with propidium iodide. Cell cycle analysis was performed using flow cytometry. LC3 staining of autophagy MCF-7 cells were seeded on cover slips 4 h prior to treatment. Cells were then treated with PQIP or AVE64 (0 l g/ml) for 4 h. Cells were fixed in 3.7% formalde- hyde and permeabilized with 0.% Triton/PBS. After blocking in % FBS/PBS, cells were stained with LC3 antibody at :50 dilution overnight, followed by incubation with secondary Alexa 488 goat anti-mouse antibody (:500) in blocking solution at RT for 30 min. Cells were mounted, and images were taken using an Olympus Fluo- view FV500 laser scanning confocal system.

500 mm 3 , mice were gavaged with or without OSI-906 (30 mg/kg). Four hours later, mice were injected with 00 l g of IGF-I intraperitoneally for 40 min then killed. Tumors were naratriptan 121679-13-8 snap-frozen, and lysates were prepared as previously described 5 . Tumor cell lysates were ana- lyzed by Western blot. Statistical analysis One-way ANOVA analysis with Tukey’s multiple com- parison test was performed in colony growth assay. If not otherwise indicated, error bars in all experiments represent standard deviation error (SE). To analyze the data in mice tumor growth experiments, individual profile plots were first generated to explore tumor growth patterns. Trans- formation on tumor volume was conducted because of presence of non-linear patterns, and tumor volume in a square root scale showed a linear time trend individually. After transformation, general linear mixed model was used to analyze longitudinal measures of tumor volume in mice where tumor volume at day 7 was the baseline 7 .

In the model, treatment, day, and treatment*day interaction were the fixed effects and random intercept and slopes were the random effects. Day was a continuous time variable, for which only linear term was considered in the model after square root transformation on tumor volume. In addition, the variance–covariance structure for two random effects was unstructured and that for random errors was first-order volunteering autoregression (AR ()). These structures were determined based on likelihood ratio test or Akaike and Bayesian informa