Figure 1
Microarray analysis of the Ser585-survival pathway. CTL-EN cells expressing either wild-type βc or the βcSer585Gly mutant were factor deprived for 18 hours and then stimulated with GM-CSF for 0, 12, and 18 hours. Total RNA was isolated, reverse transcribed, and labeled; cDNA was used to probe either NIA 15k cDNA or AMF 21K microarrays. Pairwise comparisons were performed as in panel A whereby each solid arrow represents a separate 15k cDNA slide (n = 14) and each dotted arrow represents a 21k oligonucleotide slide (n = 8) in which differential gene expression was examined in either (1) cells expressing the wild-type βc or the βcSer585Gly mutant at a single time point or (2) a single cell line at different time points. Microarray data were subjected to Bayesian analysis to identify genes that were differentially expressed in CTL-EN cells expressing the wild-type βc and βcSer585Gly mutant in response to GM-CSF at 12 hours (B) and 18 hours (C). Bayesian analysis is shown whereby the probability that a particular gene is differentially expressed (t statistic) is plotted against the magnitude (log2 scale) of the differential expression (interaction coefficient) for each gene represented on the cDNA array. Negative interaction coefficients correspond to genes that are more strongly expressed in cells expressing the wild-type βc than the βcSer585Gly mutant (eg, genes that are induced by GM-CSF in cells expressing the wild-type βc and not in the βcSer585Gly mutant). Positive interaction coefficients correspond to genes that are more strongly expressed in cells expressing the βcSer585Gly mutant than the wild-type βc (eg, genes that are repressed by GM-CSF in cells expressing the wild-type βc and not in the βcSer585Gly mutant). Selected genes identified by GO analysis in Figure 3 as having a role in cell survival or cytokine signaling and that also showed differential expression are highlighted. (D-E) Microarray data presented as the response in wild-type βc cells (M = log2 [fold change in response to GM-CSF in CTL-EN cells expressing the wild-type βc]) plotted against the response in the βcSer585Gly mutant cells (M = log2 [fold change in response to GM-CSF in CTL-EN cells expressing the βcSer585Gly mutant]). Although most genes are either not GM-CSF regulated (M values close to 0 for both wild-type βc and βcSer585Gly mutant) or are GM-CSF regulated in an equivalent manner in cells expressing the wild-type βc and βcSer585Gly mutant (M values lie close to the diagonal line), a restricted subset lies off the diagonal. The M values for the genes highlighted in panels B to E at 0, 12, and 18 hours were plotted for cells expressing either the wild-type βc (○) or the βcSer585Gly mutant (Δ) (F). (G) A clustered heat map of the 138 genes identified as being differentially expressed between the wild-type βc and βcSer585Gly mutant CTL-EN cells in response to GM-CSF was generated with the use of a Euclidean matrix. The M value (log2 fold change) for both CTL-EN cells expressing the wild-type βc or the βcSer585Gly mutant was converted to a color scale with red indicating that the gene is induced by GM-CSF, green indicating that the gene is repressed, and the color intensity indicating the magnitude of regulation.

Microarray analysis of the Ser585-survival pathway. CTL-EN cells expressing either wild-type βc or the βcSer585Gly mutant were factor deprived for 18 hours and then stimulated with GM-CSF for 0, 12, and 18 hours. Total RNA was isolated, reverse transcribed, and labeled; cDNA was used to probe either NIA 15k cDNA or AMF 21K microarrays. Pairwise comparisons were performed as in panel A whereby each solid arrow represents a separate 15k cDNA slide (n = 14) and each dotted arrow represents a 21k oligonucleotide slide (n = 8) in which differential gene expression was examined in either (1) cells expressing the wild-type βc or the βcSer585Gly mutant at a single time point or (2) a single cell line at different time points. Microarray data were subjected to Bayesian analysis to identify genes that were differentially expressed in CTL-EN cells expressing the wild-type βc and βcSer585Gly mutant in response to GM-CSF at 12 hours (B) and 18 hours (C). Bayesian analysis is shown whereby the probability that a particular gene is differentially expressed (t statistic) is plotted against the magnitude (log2 scale) of the differential expression (interaction coefficient) for each gene represented on the cDNA array. Negative interaction coefficients correspond to genes that are more strongly expressed in cells expressing the wild-type βc than the βcSer585Gly mutant (eg, genes that are induced by GM-CSF in cells expressing the wild-type βc and not in the βcSer585Gly mutant). Positive interaction coefficients correspond to genes that are more strongly expressed in cells expressing the βcSer585Gly mutant than the wild-type βc (eg, genes that are repressed by GM-CSF in cells expressing the wild-type βc and not in the βcSer585Gly mutant). Selected genes identified by GO analysis in Figure 3 as having a role in cell survival or cytokine signaling and that also showed differential expression are highlighted. (D-E) Microarray data presented as the response in wild-type βc cells (M = log2 [fold change in response to GM-CSF in CTL-EN cells expressing the wild-type βc]) plotted against the response in the βcSer585Gly mutant cells (M = log2 [fold change in response to GM-CSF in CTL-EN cells expressing the βcSer585Gly mutant]). Although most genes are either not GM-CSF regulated (M values close to 0 for both wild-type βc and βcSer585Gly mutant) or are GM-CSF regulated in an equivalent manner in cells expressing the wild-type βc and βcSer585Gly mutant (M values lie close to the diagonal line), a restricted subset lies off the diagonal. The M values for the genes highlighted in panels B to E at 0, 12, and 18 hours were plotted for cells expressing either the wild-type βc (○) or the βcSer585Gly mutant (Δ) (F). (G) A clustered heat map of the 138 genes identified as being differentially expressed between the wild-type βc and βcSer585Gly mutant CTL-EN cells in response to GM-CSF was generated with the use of a Euclidean matrix. The M value (log2 fold change) for both CTL-EN cells expressing the wild-type βc or the βcSer585Gly mutant was converted to a color scale with red indicating that the gene is induced by GM-CSF, green indicating that the gene is repressed, and the color intensity indicating the magnitude of regulation.

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