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The research protocol was conducted under French legal guidelines and approved by the local insti tutional ethics committee in accordance with Helsinki Declaration. The collection of samples is re ported to the Ministry of Education and Research No. DC 2008 purchaseABT-888 338 which is consistent with the current ethics legislation. Gene expression in breast tumors The breast tumor samples used were invasive ductal car cinoma and mostly ER positive. They were di vided into 20 SBR Grade 1, 20 SBR Grade 2, 19 SBR Grade 3, and 23 non tumor tissues. All samples used in this study were from fresh frozen tissues. The normal breast tissues were adjacent to the tumors but they are majority un matched to the tumors. Total RNA was extracted using the RNeasy Mini kit according to the manufac turers instructions, and 1 ug of total RNA was reverse transcribed with M MLV RT.<br><br> Gene expres sion was assessed by real time PCR with 4 ng of cDNA, 150 mM of primers, and 1× of iQ SYBR Green supermix from Bio Rad. Gene expression was also measured in MCF 7 cells and served to adjust the data from different plates. The data were normalized to the expression of 18S RNA and were analyzed using IQ5 software. Statistical analysis A statistical analysis Afatinib HER2 阻害剤 was performed using Students t test for most of the presented results. The values are provided as the mean standard error of the mean and were considered statistically significant at p 0. 05. The statistical analysis for the tumor samples was per formed using Minitab 16 software. The data are repre sented by box plots.<br><br> The absence of a normal distribution of each gene for each category was verified by the Anderson Darling normality test, and the non parametric Mann Whitney test was chosen to analyze our samples. Results COUP TFI overexpression modifies the basal expression of CXCL12 and CXCR4 but purchase AG-1478 not CXCR7 Our results and those of others have identified the CXCL12 CXCR4 CXCR7 axis as an important regulator of the proliferation migration balance, two mechanisms that can be modulated by COUP TFI. For this reason, we decided to investigate the impact of COUP TFI expression on the CXCL12 signaling axis in breast cancer cells. MCF 7 cells were used as an ER positive breast cancer cell model, these cells weakly express COUP TFI and represent a good model for the study of the function of COUP TFI upon overexpression.<br><br> To investigate the influence of COUP TFI on the regulation of CXCL12, CXCR4, and CXCR7 gene expression, we generated MCF 7 cell clones that stably express the full length COUP TFI tagged with an HA epitope, control cells were obtained from transfection with the 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.