Validation in liquid culture The sensitivity of strains harboring gene deletions corre sponding to proteins with functions in chromatin re modeling and transcriptional regulation was verified in liquid culture. 195 uL YPD containing 25 uM, 50 uM or 65 uM selleck kinase inhibitor CG 1521 were inoculated with 5 uL cell suspen sion. After 20 h incubation, the cell suspension was di luted 1 2 and the OD600 was measured. To normalize for differences in growth between strains the strain growth relative to wild type cells was calculated and the ratio of treated versus untreated control was determined and expressed as Net Treated Growth Value. Strains with NTGVs 0. 7 are regarded as sensitive. Strains with NTGVs 1. 2 are regarded as resistant. Exponentially growing yeast cultures were treated with 25 or 50 uM CG 1521 or vehicle control for 20 h, the OD600 was determined and the ratio was calculated.
Cell cycle analysis Yeast cells, growing in log phase, were treated with 50 uM CG 1521 or vehicle control for 1 to 4 h. After fixation in 100% ethanol for 12 16 h, cells were washed and 107 cells were resuspended in 1 mL 50 mM sodium citrate pH 7. 0 containing 100 ug mL RNase A, incubated overnight at 55 C and treated with proteinase K for 5 h at 55 C. The cells were stained with 5 uL 1 mg mL propidium iodide for 30 min and the DNA content was analyzed by flow cytometry using a BD LSR2 flow cytometer. The data were analyzed using FloJo software. Budding index analysis Flow cytometry results were confirmed by Budding Index Analysis. Exponentially growing yeast cultures were treated with 50 uM CG 1521, DMSO or 5 ug mL factor.
The budding index was cal culated by counting the number of unbudded and budded cells in approximately 100 yeast cells for each treatment condition. Cell death analysis S. cerevisiae were cultured as described above for cell cycle analysis. Aliquots were removed from the culture at 0, 2 and 4 h. The cell suspension was pelleted and re suspended in phosphate buffered saline and incubated with 1 uL propidium iodide for 10 min in the dark. PI uptake was quantitated by flow cytometry analysis using a BD LSR2 flow cytometer. The data were analyzed using FloJo software. Statistical analysis For all experiments, three or more independent bio logical replicates were performed. The results are pre sented as mean SD. Results are regarded significant if p 0.
05 as established by ANOVA and Tukey Kramer post test. Background Atherogenesis involves the cellular infiltration of several cell types, including monocytes, T lymphocytes, and mast cells. Cytokine Batimastat secretion by these cells and endothelial cells are contributing factors in the growth and propaga tion of atherosclerotic plaques as well as the stability and degradation of fibrous caps. Cytokines implicated in atherogenesis include Interleukin 1, IL 6, IL 8, IL 13, and Tumor Necrosis Factor.