control cells were obtained from transfection with the selleck Ruxolitinib empty vector. The expression of COUP TFI was first verified in the control and COUP MCF 7 cell clones. Immuno fluorescence using an antibody against the HA epitope confirmed the absence of staining in the control cells, whereas the COUP cells showed intense staining, mainly in the nucleus, corresponding to the nuclear receptor HA COUP TFI. We also confirmed these results using an anti COUP TFI antibody. As shown in Figure 1, the control cells express a low level of endogen ous COUP TFI, though COUP TFI staining is higher in the COUP cells. These results were also veri fied by western blotting. Then, the levels of CXCL12, CXCR4, and CXCR7 transcripts in the control clones and COUP clones were monitored using real time quantitative RT PCR.

Two independent control clones and two independent COUP clones were used, and the re sults shown in Figure 1B represent the mean of the data. Interestingly, the overexpression of the COUP TFI protein modified the basal expression of the CXCL12 and CXCR4 genes but did not affect CXCR7 expression. Indeed, a repression of 70% of the basal expression of CXCL12 was observed in the COUP clones compared to the control clones. In contrast, we observed a 6 fold induction of the basal expression of the CXCR4 gene. CXCR7 expression was not affected when we compared COUP clones with the control clones. These results were next confirmed at the protein level using western blotting and immunofluorescence methods.

The COUP clones dis played a striking reduction in CXCL12 protein expression, whereas the CXCR4 protein was remarkably up regulated when compared to the control clones. The CXCR7 protein did not change between the different clones. Altogether, our results suggest that COUP TFI overexpression selectively modulates the basal expression of CXCL12/CXCR4 signaling. Structural modifications at the CXCL12 and CXCR4 promoters The level of chromatin compaction appears to be well correlated with its activity, and numerous studies have reported that active transcriptional regulatory sites are present within open chromatin regions in which the nu cleosomes have been depleted. These nucleosome depleted genomic regions can be enriched from chromatin preparations using the FAIRE method. Hence, we used FAIRE to monitor the ef fect of COUP TFI on the chromatin structure of the promoters of the CXCL12, CXCR4 and CXCR7 genes in our MCF 7 clones. Interestingly, COUP TFI overexpres sion led to an 80% decrease Brefeldin_A in the amount of DNA cor responding to the open CXCL12 promoter. In contrast, the CXCR4 promoter was significantly enriched in the nucleosome depleted DNA in the cells overexpressing COUP TFI compared to the MCF 7 con trol cells.