David Schteingart (University of Michigan, Ann Arbor, www.selleckchem.com/products/Nilotinib.html MI), whereas the ST5 (14) cell line was generated and provided by Dr. Synthia Mellon (University of California, San Francisco, San Francisco, CA). Cell proliferation assay Cells were seeded on 96-well plates with increasing concentrations of the compounds added in triplicate wells. Incubation continued for 72 h (for NVP-AEW541 treatment) or 7 d (for IMC-A12 treatment) and then an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega Corp., Madison, WI) was performed according to the manufacturer��s protocol. Immunoblotting, immunohistochemistry, and RT-PCR Assays were performed as described previously (15) with primer sequences located in supplementary Table 1, published as supplemental data on The Endocrine Society��s Journals Online Web site at http://jcem.

endojournals.org. Mice Animal studies were performed in accordance to an institutionally approved protocol under the auspice of the University Committee on Use and Care of Animals at the University of Michigan. Tumor xenografts were established by sc injection of 1 �� 106 RL251 or 2.5 �� 106 H295 cells into flank regions of 4- to 6-wk-old female athymic mice. Animals were treated with IMC-A12 (8) or NVP-AEW541 (9). Mitotane was reformulated in 10% Tween 80/0.9% normal saline and administered by ip injection once daily at 300 mg/kg. DNA microarray analysis The full details of the DNA microarray analysis will be reported separately (manuscript in press).

The Affymetrix array data have already been deposited into Gene Expression Omnibus as series “type”:”entrez-geo”,”attrs”:”text”:”GSE10927″,”term_id”:”10927″GSE10927. Human adrenal tissue microarray An adrenal-specific tissue array was constructed using human formalin-fixed, paraffin-embedded tissues that correspond to the tissues used for DNA microarray analysis and was stained with either phospho-AktSer473 (Invitrogen Life Technologies, Carlsbad, CA) or phospho-IGF-1RTyr1158/62/63 (Millipore). Immunoreactivity was scored blindly by a four-tier [negative, low (1+), medium (2+), and high (3+) positive] grading scheme. Statistical analysis Tests between pairs of groups were performed with two-sided, two-sample t tests. For in vivo and in vitro data testing combinations of agents, two-way ANOVA models were used to test differences in cell viability or tumor size between difference combinations of agents and test for interactions.

We also used GSK-3 Calcusyn software to determine combination indices with in vitro mitotane and NVP-AEW541 MTS assay. Results Expression profile of IGF2 and downstream signaling in human ACC tissues Using DNA microarray technology, we analyzed human tissues derived from normal adrenal cortex, adrenocortical adenomas (ACAs), and ACCs to reveal gene expression profiles (manuscript in press). From these data, we specifically examined the 11p15.