Journal: Journal of Biological Chemistry

Article Title: Molecular Cross-talk between the TRAIL and Interferon Signaling Pathways

doi: 10.1074/jbc.m107795200

Figure Lengend Snippet: FIG. 1. Transcriptome analysis of TRAIL and interferon sig- naling pathways. Hierarchical clustering of gene expression profiles induced by death ligands and IFNs depicting 770 genes from a 9984- element cDNA microarray that are up- or down-regulated by 2-fold. The cluster diagram represents the ratio of hybridization of fluorescent cDNA probes prepared from each experimental RNA (labeled with Cy5) to the reference RNA (labeled with Cy3). These ratios are a measure of gene expression in the experimental samples (denoted as TEST) rela- tive to their specific reference sample (denoted as REF) and are de- picted according to the color scale provided. Red and green colors in the matrix represent genes that are up- and down-regulated, respectively, relative to the reference. Black color represents unchanged transcript level, while gray signifies missing data (NP, not present). Color satu- ration reflects the magnitude of the ratio relative to the median for each set of samples. Prior to hierarchical average linkage clustering, the data was filtered for a 2-fold (2.0 or 0.5) change in the Cy5/Cy3 ratio, with ratio measurements available for at least 80% of the samples. Each column in the cluster diagram represents a single hybridization exper- iment between the test and reference samples and each row represents the expression pattern of a single gene across the different experiments. This dataset along with gene names can be found in our Supplementary Material. Abbreviations used, in alphabetical order: C, cycloheximide (10 g/ml); FADDdn, FADD dominant negative MCF7 cell line; IFN, interferon; M, MCF7-pCDNA3 cells; S, SUM149 cells; Urx, untreated; Z, zVADfmk (5 M). This broad overview identifies distinct subgroups considered in detail in the text.

Article Snippet: Gene Expression Analysis—To study genes induced by activation of cell death receptors we used a 9984 element (10,000) cDNA microarray consisting of 5996 known, named genes and 2674 ESTs (Unigene Build 136) (26).

Techniques: Gene Expression, Microarray, Hybridization, Labeling, Expressing, Dominant Negative Mutation

Journal: Journal of Biological Chemistry

Article Title: Molecular Cross-talk between the TRAIL and Interferon Signaling Pathways

doi: 10.1074/jbc.m107795200

Figure Lengend Snippet: FIG. 5. Corroboration of microarray data for genes regulated by TRAIL and interferons. A, Northern blot analysis of interferon-treated samples assessed with probes against caspase 7, TRAIL, MyD88, STAT1, ISGF3, and GAPDH. GAPDH expression was used as a loading control. B, Northern blot analysis of TRAIL-treated samples assessed with probes against STAT1, ISGF3, MYD88, and IFN-R2. 28 S rRNA was used as the loading control. C, comparison between microarray data and Northern blot analyses for selected genes from A. Northern blot signal intensities were normalized against corresponding GAPDH signal intensities. The normalized values were used to calculate fold up-regulation for a given treatment using the ratio of test versus reference (for example, normalized ZC-TRAIL-24 h: normalized ZC-24 h fold up-regulation by TRAIL at the 24-h time point). Fold up-regulation as determined by Northern blot analysis was compared with fold up-regulation as obtained by microarray analysis (Cy5/Cy3 ratio of experimental with respect to reference sample). x Axes represent various treatments. y Axes on the left represent ratios obtained by Northern blot analyses and those on the right represent corresponding ratios obtained by microarray analysis. In most cases qualitative, but not necessarily quantitative, concordance between the two techniques was achieved. D, immunoblot analysis of TRAIL- and interferon-regulated proteins from MCF7 cells treated with combinations of zVAD, IFN-, or TRAIL for 24 h. Whole cell lysates were subjected to Western blot analysis using antibodies recognizing human caspase 7, TRAIL, and MyD88. Human -tubulin was used as loading control. E, MCF7 cells were cultured in serum-free medium for 2 h and then treated with CHX, TRAIL, CHX TRAIL, IFN-, or IFN- TRAIL for indicated times. Whole cell lysates were subjected to immunoblot analysis with an antibody specific for phosphorylated STAT-1 (Tyr701). Levels of STAT-1 protein were detected by stripping and reprobing of the same blot with anti-STAT1 antibody.

Article Snippet: Gene Expression Analysis—To study genes induced by activation of cell death receptors we used a 9984 element (10,000) cDNA microarray consisting of 5996 known, named genes and 2674 ESTs (Unigene Build 136) (26).

Techniques: Microarray, Northern Blot, Expressing, Control, Comparison, Western Blot, Cell Culture, Stripping Membranes