STAT3 has a well characterized role in regulating gene transcription, however, we also show through Functional Analysis, that STAT3 controls the expression of genes involved in cellular processes required to transport the proteins and regulate their subcellular localization. This supports our hypothesis that STAT3 coordinates multiple pathways within the cell and reveals that STAT3 has wide-ranging effects, controlling multiple cellular pathways involved in fundamental biological processes. Our results suggest that STAT3 orchestrates transcription, translation, transport and localization leading to wide reaching effects on cell growth, proliferation and survival. In contrast to previous studies of STAT3 target genes, we demonstrated that STAT3 regulates a diverse array of genes in both a positive and negative manner. Most genes regulated by STAT3 that have been identified to date demonstrate increased expression in cells where STAT3 is activated. However, our results also show that STAT3 signaling causes repression of many genes, including Necdin, which could profoundly impact the biology of cells harboring constitutively active STAT3. Constitutively Activated STAT3 Blocks buy AZ 960 Necdin mRNA Expression We then set out to verify the computational analysis and confirm whether Necdin is in fact a STAT3 target gene. NDN, the gene encoding Necdin, a negative growth regulator and member of the MAGE family of melanoma-associated tumor antigens, was identified as one candidate STAT3-regulated gene. Five Affymetrix probesets corresponding to NDN are ranked in the list of the top 12 most significantly repressed probesets . When compared with normal control cells, analysis of the microarray data demonstrated that NDN expression was consistently repressed in the cell lines expressing v-Src or STAT3-C, indicating that NDN is a candidate STAT3-regulated gene in both of these cell lines . Figure 1B. confirms that NDN mRNA expression is dramatically down-regulated in v-Src and STAT3-C expressing cells as measured by quantitative Real-Time PCR. NIH-3T3 cells stably expressing v-Src express high levels of active STAT3. These v-Src 3T3 cells were treated with either control siRNA or two different doses of STAT3-specific siRNA. Cells treated with control siRNA maintain high levels of STAT3 and have low levels of Necdin expression . As expected, STAT3 siRNA effectively inhibited expression of total STAT3 .