Expression of the two CXCL12 receptors in NB cell lines.

<p>(A) Qualitative RT-PCR analyses for <i>CXCR7</i> and <i>CXCR4</i> mRNA expression in a panel of N-, I-and S-type NB cell lines. <i>GAPDH</i> was used as gene of reference. The prostate cancer cell line PC-3 and the breast cancer cell line MCF-7 were used as positive controls for <i>CXCR7</i> expression. (B) Flow cytometry analyses of CXCR7 and CXCR4 cell surface expression in NB cell lines. Percent represents CXCR7-or CXCR4-positive cells. Grey line: cells stained without the primary Ab. Black line: cells stained with anti-CXCR7 or anti-CXCR4. (C) Semi-quantitative real-time PCR analyses of <i>CXCR7</i> mRNA expression level in the IGR-NB8 cell line after 3 days of treatment with either 10 µM RA or BrdU (left panel). Expression levels of <i>CXCR7</i> transcripts were calculated relatively to the level of the housekeeping gene <i>HPRTI</i>. Untreated (unt.) cells and DMSO-treated NB cells represented control conditions. Columns indicate results in triplicates, and were representative of two independent experiments. Error bars indicate S.D. Student’s t-test: *p<0.05, **p<0.01. Images (right panel) represent immunofluoresence staining of β₃-tubulin (red) and DAPI (blue) in treated or untreated IGR-NB8 cells. (D) CXCR7, CXCR4, or a combination of the two CXCL12 receptors was overexpressed in the IGR-NB8 and the SH-SY5Y cell lines. As all vectors encoded for EGFP, transduced GFP-expressing NB cells were sorted by FACS to control transfection efficiency. Both NB8pMigr and SHSYpMigr cell lines represented control cells, transduced with the pMigr empty vector. Percent of CXCR7-and CXCR4-positive transduced cells are indicated as well as the mean fluorescent intensity (brackets) for CXCR7 and CXCR4 staining. Dark and grey lines: cells stained without anti-CXCR7 and anti-CXCR4, respectively; Green and blue lines: cells stained with anti-CXCR7 and anti-CXCR4, respectively. (E) Semi-quantitative real-time PCR analyses for <i>CXCR7</i> mRNA expression levels in NB transduced cell lines. Experiment was performed in triplicates. Error bars indicate S.D.