For measurement of cardiac valve size and calcification, serial sagittal sections were collected from each treatment group. Von Kossa,s Camptothecin Topoisomerase inhibitor stain was used as a marker to visualize calcification. Gene expression Total RNA was extracted from the lower half of the LV from B6 wild type mice using TRIzol. After DNAse treatment, 500 ng of total RNA was reverse transcribed using the High Capacity cDNA Archive Kit. The expression of hypertrophy markers atrial natriuretic peptide and brain natriuretic peptide, pro and antiapoptotic markers and ERBB receptors and ligands was determined by real time quantitative PCR using Taqman Univeral Master Mix and Assays on Demand primers and probes. Results are represented as mean fold changes relative to control groups. Reactions were run on a Stratagene MX3000P machine with analysis software.
Threshold cycles were determined by an in program algorithm assigning a fluorescence baseline based on readings prior to exponential amplification. Fold change in expression was calculated using the 2 ΔΔcT method, with Actb or Gusb as endogenous controls. In vivo phosphorylation assays B6 wild type male mice maintained on control or experimental diet for 90 days buy Chrysin were injected subcutaneously with 5 g/g body weight of EGF in PBS. After 10 minutes, mice were euthanized and livers and hearts removed, frozen in liquid nitrogen and stored at 80. The frozen tissues were sonicated in 5 10 ml/g tissue of lysis buffer consisting of 20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X 100, 1 mM PMSF, 10g/ml of leupeptin, 10 g/ml of aprotinin, 1 mM sodium vanadate and 10 mM glycerophosphate at 4.
The tissue lysates were cleared by centrifugation for 10 min at 4 and protein concentrations were determined by the Bradford assay. Protein lysates were separated by denaturing 7.5% sodium dodecylsulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes. Protein blots were incubated overnight at 4 with rabbit polyclonal antibodies recognizing EGFR, phospho EGFR or phospho p44/42 MAP Kinase followed by incubation Barrick et al. Page 4 Toxicol Appl Pharmacol. Author manuscript, available in PMC 2009 May 18. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript with goat anti rabbit horseradish peroxidase conjugated antibody and detection with an enhanced chemiluminesence system.
Statistical analysis Data is presented as mean / standard error of the mean. Data from control groups was pooled when there was no significant difference between parameters. The non parametric Wilcoxon rank sum test was used to compare tumor number and size between treatment groups. The Mann Whitney or unpaired Student,s t test was used to compare data between respective treatment and control groups. The Kruskal Wallis test or analysis of variance was used to detect significance by treatment. All analyses were performed using StatView software. A p 0.05 is considered statistically significant. Results Orally delivered AG 1478 is biologically active Although the reversible EGFR inhibitor AG 1478 has been used extensively in numerous in vitro and in vivo studies, to our knowledge it has not previously been shown to have activity when delivered orally. Pharmacokinetic studies in wild type mice using 3H AG 1478 showed that tissue distribution is highest in liver, which also has the highest total and phospho EGFR protein conten