The genes related to multiple signal pathways such as cell cycle control and apoptosis were presented. The profound changes in gene biological activity expression elucidate the molec ular basis for the pleiotropic effects of butyrate on biolog ical processes. The results presented in this paper can provide clues on the mechanism of histone deacetylase inhibition by butyrate and resulting alterations in the expression of genes involved in cell cycle, apoptosis, and transcriptional regulation. Since butyrate functions as both a nutrient and signaling molecule regulating the cell growth and proliferation, these findings enable better rec ognition of the full range of roles butyrate may play dur ing cattle energy metabolism, cell growth and proliferation.

Methods Cell Culture and Treatments The Madin Darby bovine kidney epithelial cells were cultured in Eagles minimal essen tial medium supplemented with 5% fetal bovine serum in 25 cm2 flasks with medium renewal twice per week. Cell cultures were maintained in a water jacked incubator with 5% CO2 at 37 C. Sub culti vations were performed when cells attained 80 to 90 % confluence, according to the product information sup plied by American Type Culture Collection. At approxi mately 50% confluence, the cells were treated with 10 mM of sodium butyrate for 24 h were used for the flow cytometry and microarray experiments. The harvested cells were snap frozen in liq uid N2 and stored at 80 C until RNA extraction. Flow Cytometric Analysis of Cells The detailed procedures were described in a previous pub lication.

Briefly, cells collected by trypsinization were washed and resuspended in PBS buffer. Two volumes of ice cold 100% ethanol were added drop wise into tubes and mixed with cells in suspension by slow vertexing. After incubation with RNase Brefeldin_A I, cells were then stained with propidium iodide. Measuring the fluorescence by flow cytometry provided a measure of the amount of PI taken up by the cells and, indirectly, the amount of DNA content. Cell DNA content was analyzed using a flow cytometer and collected data were analyzed using Cytomics RXP. At least 10,000 cells per sample were ana lyzed. Preparation of Cell Extracts and Western Blot Analysis Preparations of cells and cell extracts, SDS PAGE and Western Blot analysis were described previously. Briefly, the protein from different samples was separated by SDS PAGE on two identical 4 to 20% polyacrylamide gradient gels. One gel was stained with SimpleBlue and one was transferred to a membrane and probed with monoclonal anti acetyl phospho H3 and anti acetyl H3 antibodies. The target bands on the West ern Blots from three experiments were quantified with a NIH Image software. The relative densities were measured and corrected with the stained protein density.