At the time of sacrifice, intestinal sections were collected and flushed with ice-cold phosphate buffered saline. The duodenal and jejunal sections were cut longitudinally, and the epithelium was scraped using disposable sterile plastic spatulas (VWR International) into vials containing ~ 1 ml of TRIzol (Invitrogen, Carlsbad, CA) and snap-frozen

in liquid nitrogen. The samples were stored at − 80 °C and shipped overnight on Selleck BTK inhibitor dry ice to Michigan State University for gene expression analysis. All procedures were carried out with the approval of the Institutional Animal Care and Use Committee at Southern Research Institute. Frozen intestinal epithelial samples were homogenized using a Mixer Mill 300 tissue homogenizer (Retsch, Germany). Total RNA was isolated according to the manufacturer’s protocol with an additional acid phenol:chloroform extraction. Isolated RNA was resuspended in RNA storage solution (Ambion Inc., Austin, TX), quantified (A260), and quality was assessed by evaluation of the A260/A280 ratio and by visual inspection of 1 μg total RNA on a denaturing gel. Dose-dependent changes in gene expression were examined using mouse 4 × 44 K Agilent whole-genome

oligonucleotide microarrays (version 1, Agilent Technologies, Inc., Santa Clara, CA). Treated samples were co-hybridized with vehicle controls to individual arrays according to the manufacturer’s protocol (Agilent Manual: G4140-90050 v. 5.0.1). All hybridizations were performed with three independent Ganetespib biological replicates for treated and control tissues (i.e., RNA samples were not pooled) and independent labeling of each sample (Cy3 and Cy5, including dye swap) for each treatment group at each time point (8 and 91 days). Microarray slides were scanned at 532 nm (Cy3) and 635 nm (Cy5) on a GenePix 4000B scanner (Molecular Devices, Union City, CA). Images were analyzed for feature and background intensities using GenePix Pro 6.0 software (Molecular Devices). All data passed our laboratory quality assurance protocol (Burgoon et al., 2005) and were deposited in TIMS dbZach data management system (Burgoon and Zacharewski, 2007). Microarray

experimental design is shown in Supplementary Fig. S1. Microarray data were normalized using a semi-parametric approach (Eckel et al., 2005). The posterior probabilities were Dichloromethane dehalogenase calculated using an empirical Bayes method based on a per gene and dose basis using model-based t-values ( Eckel et al., 2004). Unless stated otherwise, gene expression data were ranked and prioritized using |fold change| > 1.5 and statistical P1(t) value > 0.999 criteria to identify differentially expressed genes. P1(t) values represent the posterior probability of gene activity on a per gene and treatment dose basis using the model-based t-value ( Eckel et al., 2004). Annotation and functional categorization of differentially regulated genes was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID) (Dennis et al.