Ession data analysis (Rosetta Biosoftware, Seattle, WA, USA). The intensity ratiosEssion data analysis (Rosetta Biosoftware,

Ession data analysis (Rosetta Biosoftware, Seattle, WA, USA). The intensity ratios
Ession data analysis (Rosetta Biosoftware, Seattle, WA, USA). The intensity ratios between the cell line sample and mixed reference calculated for each sequence were computed JNJ-26481585 site according to the Agilent error model. A particular sequence was considered differentially expressed if the calculated p-value of change was P 0.01. These data are available with accession number [GEO:GSE18496].Proliferation assays Cells were seeded in duplicate at 5,000 to 10,000 cells per well in 24-well plates. The day after plating, PD 0332991 was added at 1 M and twofold dilutions over six concentrations were performed to generate a dose-response curve. Control wells without drug were also seeded. Cells were counted on day 1 when the drug was added as well as after 6 days when the experiment ended. After trypsinization, cells were placed in Isotone solution and counted immediately using a Coulter Z2 particle counter (Beckman Coulter Inc., Fullerton, CA, USA).For each assay, the log of the fraction growth inhibition was plotted against the log of drug concentration, and the linear regression curve fit correlation coefficient (r value) was calculated. Multiple drug effect analysis was performed using computer software as previously described [33]. Combination index (CI) values were derived from parameters of the median effects plots, and statistical tests were applied (unpaired, two-tail Student t test) to determine whether the mean CI values at multiple concentrations were significantly different from CI = 1. In this analysis, synergy is defined as CI values significantly lower than 1.0, antagonism as CI values significantly higher than 1.0, and additivity as CI values equal to 1.0. All CI values were calculated using the conservative assumption of mutually nonexclusive drug interactions. All experiments were carried out at least twice. Combination studies were performed as above with the MCF7 tamoxifen-resistant clones with addition of estrogen back to the media at the time of the experiment (as described in cell culture above).Western blot analysis Cells in log-phase growth were treated with 100 nM PD 0332991 and were harvested at various timepoints by washing in PBS and lysis at 4 in RIPA lysis buffer. Insoluble material was cleared by centrifugation at 10,000 ?g for 10 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27196668 minutes and protein was quantitated using bicinchoninic (BCA) (Pierce Biochemicals, Rockford, IL, USA). Protein content was resolved by SDS-PAGE electrophoresis, and was transferredPage 3 of(page number not for citation purposes)Breast Cancer ResearchVol 11 NoFinn et al.to nitrocellulose membranes (Invitrogen). Total pRb expression was detected using a rabbit polyclonal antibody to pRb (Abcam, Cambridge, MA, USA). Rb phosphorylation was detected using rabbit polyclonal antibody to phospho-serine 780 (Cell-signaling, Danvers, MA, USA). Blots were washed and incubated with a goat-anti-rabbit IgG horseradish peroxidase conjugate (Upstate, Bellerica, MA, USA), developed using ECL Plus chemifluorescent reagent (Amersham Biosciences, Pistcataway, NJ), and imaged using chemifluorescence.Cell cycle analysis and apoptosis studies Effects of PD 0332991 on the cell cycle were assessed using Nim-DAPI staining (NPE Systems, Pembroke Pines, FL, USA). Cells were plated evenly in control and experimental wells, were allowed to grow to log phase and were then treated with 100 nM PD 0332991 for the defined times. To perform cell cycle analysis, cells were washed with PBS; then trypsin was applied to release c.