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. Author manuscript; available in PMC: 2009 Aug 15.

Published in final edited form as: Toxicol Appl Pharmacol. 2008 Apr 27;231(1):77–84. doi: 10.1016/j.taap.2008.04.014

A) Immunohistochemical detection and quantitation of expression or phosphorylation of RAS downstream effectors. Statistical evaluation of the immunohistochemical determination of phospho-p53, p19, caspase-3, phospho-AKT, survivin, and phospho-JNK in lung tumor tissue from bitransgenic mice treated with 25, 100, or 500 μg/ml of DOX relative to the surrounding benign tissue. A mixed model approach was utilized to determine the signal intensity; results are expressed as the mean cell counts ± the standard error. *, denotes p-values b etween the normal and overall carcinogenic tissue was significant at ≤.05. B) Representative IHC slides showing staining with antibodies against JNK, p53, p38, and Ki-67 in surrounding normal tissue and lesions from mice treated with 25, 100, or 500 μg/ml of DOX; 20-40x magnification, images captured with the Image J software. C) Determination of mRNA expression of p19^ARF and survivin. The levels of expression of p19^ARFand survivin were determined by real-time fluorescent RT-PCR in normal lung tissue and adenomas from Ki-ras^G12C mice treated with 0, 25, 100, or 500 μg/ml of DOX. Each gene value is expressed as the average copy number ± SE and is corrected for the level of expression of GAPDH. Statistical differences in gene expression were determined by comparing samples from treated bitrangenic mice at each of the 3 doses to untreated bitransgenic mice (control samples). **, denotes significant difference in gene expression between treated tissue and control tissue with p value ≤ 0.05. *, denotes marginally significant difference in gene expression between treated tissue and control tissue with p value = 0.08.

A) Immunohistochemical detection and quantitation of expression or phosphorylation of RAS downstream effectors. Statistical evaluation of the immunohistochemical determination of phospho-p53, p19, caspase-3, phospho-AKT, survivin, and phospho-JNK in lung tumor tissue from bitransgenic mice treated with 25, 100, or 500 μg/ml of DOX relative to the surrounding benign tissue. A mixed model approach was utilized to determine the signal intensity; results are expressed as the mean cell counts ± the standard error. *, denotes p-values b etween the normal and overall carcinogenic tissue was significant at ≤.05. B) Representative IHC slides showing staining with antibodies against JNK, p53, p38, and Ki-67 in surrounding normal tissue and lesions from mice treated with 25, 100, or 500 μg/ml of DOX; 20-40x magnification, images captured with the Image J software. C) Determination of mRNA expression of p19^ARF and survivin. The levels of expression of p19^ARFand survivin were determined by real-time fluorescent RT-PCR in normal lung tissue and adenomas from Ki-ras^G12C mice treated with 0, 25, 100, or 500 μg/ml of DOX. Each gene value is expressed as the average copy number ± SE and is corrected for the level of expression of GAPDH. Statistical differences in gene expression were determined by comparing samples from treated bitrangenic mice at each of the 3 doses to untreated bitransgenic mice (control samples). **, denotes significant difference in gene expression between treated tissue and control tissue with p value ≤ 0.05. *, denotes marginally significant difference in gene expression between treated tissue and control tissue with p value = 0.08.