The compound showed a negligible effect on the metabolism of other agents and i

The antibodies used for the Western blot assays were diluted 1,2000 or 1,5000. The supplier ABT-888 detection of the immunocomplexes was performed using an enhanced chemiluminescence system. For the detection of ERK ac tivity, control and COUP cells were cultured for 48 h in phenol red free DMEM with 0. 5% dsFBS. After EGF or CXCL12 stimulation, whole cell extracts were directly prepared in 3× Laemmli buffer. Following sonication, the protein ex tracts were denatured for 5 min at 95 C and analyzed as detailed above. Formaldehyde assisted Isolation of Regulatory Elements FAIRE was performed as described by Eeckhoute et al. Briefly, asynchronously growing MCF 7 cells treated or not for 48 h with 10−8 M E2 were cross linked with 1% formaldehyde for 10 min at room temperature.<br><br> Glycine was added to a final concen tration of 125 mM, and the cells were rinsed with cold PBS and harvested. The cells were lysed with a solution of 1% SDS, 10 mM EDTA, and 50 mM Tris HCl containing a protease purchaseAfatinib inhibitor cocktail and then sonicated for 14 min using a Bioruptor set at the highest inten sity. The soluble chromatin was subjected to three con secutive phenol chloroform extractions and incubated overnight at 65 C to reverse the cross linking. The DNA was then purified using the MinElute PCR purification kit. The relative enrichment of open chromatin for the CXCL12, CXCR4 and CXCR7 genes was quantified by real time PCR performed using the iQ SybrGreen supermix and a Bio Rad MyiQ appar atus. The primers used for the quantitative PCR experi ments were described previously.<br><br> Proliferation assay A total of 2500 MCF 7 cells clones per well were seeded in 96 well plates and cultured in 100 supplier AG-1478 uL of phenol red free DMEM 2. 5% dsFBS and EtOH or CXCL12 for 7 days. Every 2 days, the medium was removed, and fresh treatments were performed. Proliferation was evaluated using the 3 2,5 diphenyltetrazolium bromide assay. 10 uL of 5 mg mL MTT solution was added to 100 uL of cul ture medium in each well and incubated for 2 h at 37 C. The supernatant was removed, and the formazan formed was dissolved in 100 uL DMSO. The absorbance of each well at 570 nm was measured using a microplate reader. Migration assay The cells were cultured for 48 h in phenol red free DMEM with 5% dsFBS prior to the experiments. A total of 50,000 cells were plated in the upper chamber of a BDBiocoat control insert in phenol red free DMEM 0.<br><br> 5% dsFBS with or without AMD3100 or CXCL12, phenol red free DMEM 2. 5% dsFBS with or without AMD3100 or CXCL12 was added to the lower chamber. The cells were allowed to migrate for 24 h at 37 C, and the non migrant cells were wiped off the upper chamber with a cotton swab. The insert was then placed in phenol red free DMEM 2. 5% dsFBS with calcein AM for 1 h to stain the cells that reached the lower side of the fil ter. The migrant cells were then counted in 3 fields from at least 3 inserts per experimental condition. Ethics statement Human samples were obtained from the processing of biological samples through the Centre de Ressources Biologiques Santé of Rennes. We have received written informed consent from all patients for the use of their samples analyzed in this study.