Journal:

Article Title: Effects of Mutant Human Ki- ras G12C Gene Dosage on Murine Lung Tumorigenesis and Signaling to its Downstream Effectors

doi: 10.1016/j.taap.2008.04.014

Figure Lengend Snippet: 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 p19ARF and survivin. The levels of expression of p19ARFand survivin were determined by real-time fluorescent RT-PCR in normal lung tissue and adenomas from Ki-rasG12C 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.

Article Snippet: The following primary antibodies were used: anti-cyclin D1 1:100 (Abcam Inc., Cambridge, MA); anti-p19 ARF 1:100 (Abcam Inc. Cambridge, MA ); anti-survivin 1:500 (Abcam Inc., Cambridge, MA); anti-phospho- p53 (Ser 389 ) 1:50 (Cell Signaling Technology); anti-caspase-3 (Cell Signaling Technology, Beverly, MA); anti-phospho-SAPK/JNK (Cell Signaling Technology}, anti-phospho-p44/42 MAPK (Thr 202 /Tyr 204 ) 1:60 (Cell Signaling Technology Beverly, MA); anti-phospho-p38 MAPK (Thr 180 /Tyr 182 ; 12F) 1:100 (Cell Signaling Technology Beverly, MA), anti-phospho-AKT (Ser 473 ; 736E11) 1:50 (Cell Signaling Technology, Beverly, MD); and anti-Ki-67 (Abcam Inc. Cambridge, MA) Samples as well as negative controls (which lacked the primary antibody) were incubated overnight at 4°C.

Techniques: Immunohistochemical staining, Quantitation Assay, Expressing, Phospho-proteomics, Staining, Software, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control