Supplemental Materials and Methods

Transcription

1 Supplemental Materials and Methods 125 I-CXCL12 binding assay KG1 cells ( ) were preincubated on ice with cold CXCL12 (1.6µg/mL corresponding to 200nM), CXCL11 (1.66µg/mL corresponding to 200nM), anti-cxcr7 antibody clone 11G8 (10µg/mL) or CCX771 small-molecule compound (1µM) in RPMI, BSA 1%, HEPES 20 mm, ph 7.4 for 45 min. 125 I-CXCL12 (1.6ng/mL corresponding to 0.2nM) was then added for 3h at 37 C. Non-specific internalized ligand was determined by maintaining the cells on ice throughout the experiment. KG1 cells were then incubated for 5 min with stripping buffer composed of 0.5 M NaCl, 0.2 M Acetic Acid, ph 2.6. The mixture was added on a 12% sucrose cushion and centrifugated for 10 min. Supernatant containing the unbound 125 I- CXCL12 was aspirated and the cell pellet was resuspended and transferred into a fresh counting tube. 125 I-CXCL12 was quantified by Gamma counter Packard Cobra II Auto- Gamma. Expression of a CXCR7-YFP plasmid CXCR7-YFP plasmid, a kind gift from r A. Levoye and r F. Bachelerie (Institut Pasteur, Paris, France) (Levoye et al. Blood., 2009, 113(24): ) was transfected in SkBr3 cells with FuGENE H transfection reagent for 48h following FuGENE H protocol database ( SkBr3 cells were then stained with anti-cxcr7 antibody and analyzed by B Fortessa flow cytometer and TC5 SP5 confocal microscope (Leica Wetzlar-Germany).

2 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Reverse transcription of total RNA from C34 cells was achieved using TaqMan Reverse Transcription Reagents (Applied Biosytems) as previously described (Chabanon et al., Stem Cells 2008, 26(12): ). For not quantitative PCR, cna was amplified for 40 cycles using HotStart Taq polymerase kit (Quiagen). Specific Primer sequences are detailed in Table S2.

3

4 I-CXCL12 (in percentage) 50 0 CTL CXCL12 CXCL11 anti-cxcr7 (11G8) CCX771 Supplemental Figure 2: 125 I-CXCL12 binds to CXCR7 in C34 KG1 cells C34 KG1 cells were pre-incubated on ice for 45 min with medium alone (CTL) or with CXCL12, CXCL11, anti-cxcr7 antibody (clone 11G8) or the CCX771 small-molecule antagonist. 125 I-CXCL12 was then added and cells incubated for 3h at 37 C to promote endocytosis of bound radioligand. Cells were then put back on ice and surface-bound radioligand removed by incubation with an acidic stripping buffer. Cell associated (internalized) radioligand was separated from unbound by centrifugation through a 12% sucrose cushion. Histograms show cell-associated 125 I-CXCL12; data are expressed as the mean percentage ± S (n³5) of the value measured in control cells. indicate the significance of differences between experimental conditions (ANOVA, p<0.001).

5 Cyclin1 Fold change vs. isotype control G 0 G 1 SG 2 /M CyclinA Fold change vs. isotype control CyclinB1 G 0 G 1 SG 2 /M Fold change vs. isotype control G 0 G 1 SG 2 /M SF-1 anti-cxcr7 anti-cxcr4 Supplemental Figure 3: CXCR7 participates with CXCR4 in CXCL12-induced cyclin1, A, B1 overexpression in PB C34 cells PB C34 cells were stimulated 48 hours in a serum- and cytokine-free StemaA medium in the presence or absence of CXCL12 (0.5 ng/ml), neutralizing anti-cxcr4 and/or -CXCR7 antibodies or isotype control. After Cyclin 1, A, B1/PI labeling, the percentages of cyclin 1, A, B1 expressing cells in G 0 /G 1 or SG 2 /M phases were determined by flow cytometry. Histograms show the modulations of the percentages of cyclin 1, A, B1 expressing cells in G 0 /G 1 or SG 2 /M phases vs. control cells as mean of fold changes ± S (n>3)., indicate significance versus control cells (p 0.05 and p 0.01)., indicate significance between conditions (p 0.05 and p 0.01).

6 SUPPLEMENTAL TABLES Table S1 : Antibodies Antibody Manufacturer Clone CXCR4 R&Systems 12G5 CXCR4 Abcam CXCR4-FITC R&Systems 12G5 CXCR4-PE B Biosciences 12G5 CXCR7 MBL 9C4 CXCR7 ChemoCentryx Inc. 11G8 CXCR7 GeneTex CXCR7-PE MBL International 9C4 C34-Percp B Biosciences 8G12 β-arrestin2 Cell Signaling C169 Phospho-Akt Cell Signaling 193H12 Akt Cell Signaling Actin MBL 61 Ki67-FITC B-Pharmingen B56 Cyclin A-FITC B-Pharmingen BF683 Cyclin B1-FITC B-Pharmingen GNS-1 Cyclin 1-FITC B-Pharmingen G Cyclin 3-FITC B-Pharmingen G IgG1 B Biosciences MOPC-21 IgG2a R&Systems IgG1-PE B Biosciences MOPC-31C IgG1-Percp B Biosciences X40 IgG2a-FITC Immunotech U7.27 IgG2a-PE B Biosciences G

7 Table S2 : Primers sequences Name CXCR4 CXCR7 Cyclin1 Cyclin3 CyclinE1 P27 TBP Sequence Forward 5 CCCATCCTCTATGCTTTCCTTG3 Reverse 5 GTCCACCTCGCTTTCCTTTG3 Forward 5 TGCTCCCTCTTCACTCTACTC3 Reverse 5 TCTGGGACGGGTTTACTTGTT3 Forward 5 TTCTTATCCCCTGCCCCTTC3 Reverse 5 CACTCTTGCCACCTCCCTTC3 Forward 5 CCTGGATCGCTACCTGTCTTG3 Reverse 5 CAGCGTGGTCGGTGTAGATG3 Forward 5 CCTGGATGTTGACTGCCTTG3 Reverse 5 TATGTCGCACCACTGATACCC3 Forward 5 AAAGCAACAGAAACCTATCCTCAC3 Reverse 5 AACATTCAAAACTCCCAAGCAC3 Forward 5 TGGTGTTGTGAGAAGATGGATG3 Reverse 5 CCAGATAGCAGCACGGTATGAG3