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Supplementary Material
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A) Methods:
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Quantitative RT-PCR
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mRNA expression was determined by quantitative real-time PCR (RT-PCR) using SYBR Green.
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One microgram of total mRNA, which was isolated using the RNeasy kit (Qiagen), was used for
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cDNA synthesis using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems),
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and 50 ng of cDNA was used for RT-PCR using the Power SYBR Green PCR Mastermix
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(Applied Biosystems) on an ABI Prism 7700 sequence detection system (Applied Biosystems).
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Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
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mRNA using the ∆∆Ct-method. The oligonucleotides used are atctgtcatctgcctcactgacg (CXCR4-
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forward), aaggtggtctatgttggcgtctg (CXCR4-reverse), gtggtctccctgactttcaacagc (GAPDH-forward)
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atgaggtccaccacctgcttgctg (GAPDH-reverse).
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CXCR4 surface expression
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Cell surface expression of CXCR4 was analyzed by flow cytometry after staining with APC-
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conjugated anti-human CD184 (12G5) antibody (BD Pharmingen). Non-specific binding was
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determined by using the APC-conjugated rat IgG2a as isotype control (BD Pharmingen).
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CXCR4 internalization and recycling assay
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Cells were plated in starving medium (RPMI containing 2% FCS) at 5x104/mL for two hours. For
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internalization assay, cells were then incubated for 30 or 60 min with CXCL12 (0-100nM) at
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37ºC. For recycling assay, cells were incubated for 30 min with CXCL12 (50nM) at 37ºC. Half of
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the cells were washed three times in PBS to remove CXCL12 and resuspended in new starving
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medium. Cells were collected at indicated time points and stained with anti-human CD184
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antibody or isotype for 45 min at 4°C before to be fixed with 4% PFA and analyzed by flow
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cytometry.
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Confocal microscopy analysis
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HEK-293 cells were cultured on a glass slide and stained with PHK-26 when indicated before to
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be fixed with 4% paraformaldehyde for 10 min at 37°C and washed with PBS. DAPI was used
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for DNA staining. Slides were washed and mounted with Fluorsafe Reagent (Calbiochem).
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Confocal microscopy is carried on a LSM710 laser-scanning microscope with a 63x objective
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(Zeiss) and image analysis was performed using Zen 2010 software (Zeiss). The CXCR4
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surface expression was determined as the surface area coverage of the specific GFP signal that
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do not co-localize with PHK-26 signal per region of interest selected based on an equivalent
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area of cytoplasm and membrane in 18 microscopic fields.
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Immunoblotting
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Cells were lysed in lysis buffer [10 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% Triton X-
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100, 0.5 mmol/L EDTA, 10% glycerol, 10 mmol/L NaF, 1 mmol/L Na3VO4], supplemented with
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protease inhibitor cocktail (Calbiochem) for 20 min on ice followed by centrifugation at 12 000×g
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for 15 min. Cleared lysates were assayed for protein concentration using the Bradford protein
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assay system (Bio-Rad). 50 µg of protein were loaded on a 12% SDS-PAGE and transferred on
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a nitrocellulose membrane. Immunoblotting was performed using anti-Bax (1:500) (Cell
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Signaling), anti-Bak (1:500) (D4E4, Cell signaling), anti-Bcl-2 (1:200) (50E3, Cell signaling), anti-
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Bcl-xL (1:500) (54H6, Cell signaling) or anti-β-Actin (1:5000) (AC-74; Sigma); the latter was used
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as a reference. Bound primary antibodies were detected with either horseradish peroxidase-goat
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anti-rabbit antibody or horseradish peroxidase-goat anti-mouse antibody (Thermo Scientific).
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Enhanced chemiluminescence Western blotting detection was performed using West Femto
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SuperSignal reagent (Thermo Scientific).
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ERK phosphorylation measurement
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Cells were plated in starving medium (RPMI containing 2% FCS) at a concentration of
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0.5x105/mL for two hours. Cells were then incubated with CXCL12 (50nM) at 37ºC for the
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indicated time. Cells were then fixed with 4% PFA at 37°C for 10 min. After washing with cold
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PBS, cells were resuspended 30 min in 90% ice-cold methanol. Permeabilized cells were
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stained at room temperature with anti-phospho-ERK (Thr202/Tyr204) (Cell Signaling) followed
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by Alexa Fluor 647 conjugated goat anti-rabbit IgG (Invitrogen) and washed with PBS before
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analysis by flow cytometry.
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Determination of intracellular Ca2+ flux
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Cells were plated in starving medium (RPMI containing 2% FCS) at a concentration of
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0.5x105/mL for two hours. After washing, cells were then loaded with 1 µM Indo-2-AM (Molecular
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Probes) in calcium buffer (137mM NaCl, 2.7mM KCl, 1.8mM CaCl2, 1mM MgCl2, 5.6mM
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Glucose, 0.1%BSA and buffered with 20 mM HEPES at pH 7.4). After incubation at 37°C for 30
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min, cells were washed twice and resuspended in calcium buffer to a final concentration of 2x106
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cells/ml. Indo-2 fluorescence was monitored in an LS50B luminescence spectrophotometer
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(Perkin-Elmer).
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Flow cytometric immunophenotyping
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Cell surface expression of CXCR7, CD34, CD49d, CD49e and CD62L was analyzed by flow
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cytometry after staining with APC-conjugated anti-human CXCR7 (11G8) antibody (R&D
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systems), PE-conjugated anti-human CD34 (581) antibody (BD Pharmingen), PE-conjugated
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anti-human CD49d (9F10) antibody (BD Pharmingen), PE-conjugated anti-human CD49e (IIA1)
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antibody (BD Pharmingen), PE-conjugated anti-human CD62L (SK11) antibody (BD
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Pharmingen). Non-specific binding was determined by using the APC- or PE-conjugated rat
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IgG2a as isotype control (BD Pharmingen).
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Statistical analysis
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For clinical data, statistical analyses, including data description, were performed using the
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Statistical Package of Social Sciences version 19.0 for Windows (SPSS, Chicago, IL).
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Spearman test was used to analyse relationships between the markers, and only correlations
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with a >0.2 or <-0.2 were further considered. The prognostic performance of the variables and
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determination of optimal cut-off values were established by receiver operating characteristic
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(ROC)-curves plotting sensitivity vs 1-specificity with special considerations of the respective
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area under the ROC (AUROC). The Youden’s index (Y = sensitivity + specificity – 1) was used to
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determine optimal cut-off with the highest sensitivity and specificity when there is no particular
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requirement on sensitivity and/or specificity (20, S1). Then, survival was analysed using the
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Kaplan-Meier method and compared using the log-rank test.
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Statistical significance was defined as p<0.05, and two-sided tests were used throughout. Data
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is represented as the mean  standard error of the mean (SEM). Comparisons between all
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groups were performed using the Kruskal-Wallis, Mann-Whitney or ANOVA tests and only
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significant differences are presented. Statistical analyses were performed using GraphPad Prism
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(Graph Pad Software, La Jolla, CA, USA).
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S1.
Tzankov A, Zlobec I, Went P, Robl H, Hoeller S, Dirnhofer S. Prognostic
immunophenotypic biomarker studies in diffuse large B cell lymphoma with special
emphasis on rational determination of cut-off scores. Leuk Lymphoma 2010 Feb; 51(2):
199-212.
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B) Figures:
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Figure S1: Survival of AML patients according to pCXCR4 expression in BM samples
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determined by IHC. Kaplan-Meyer estimates of overall survival according to pCXCR4
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expression of different risk groups of AML patients.
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Figure S2: Expression of CXCR4 variants. (a) CXCR4 mRNA expression in Kasumi-1
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wildtype and expressing the different variant of the receptor measured by quantitative RT-PCR.
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Results were normalized to GAPDH levels. Fold expression relative to Jurkat cells are
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represented (data represent mean  SEM values of 4 independent experiments performed in
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duplicates). (b) Confocal immunofluorescence images of the Mock, CXCR4-WT, -S339A or -
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S339E fused to GFP (green) localization in HEK-293 cells. Hoechst was used to stain nuclei
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(blue).
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Figure S3: Role of CXCR4 variants in internalization and recycling. (a) Confocal
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immunofluorescent localization of the CXCR4-WT, -S339A or -S339E fused to GFP before
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(control) after CXCL12 stimulation and +/- wash-out at 30min on HEK-293 cells. Hoechst was
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used to stain nuclei (blue). A representative maximal projection of Z-stack acquired cells out of
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18 fields is shown. (b) Relative surface CXCR4 expression on HEK-293 cells determined after
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CXCL12 stimulation +/- wash-out at 30 min expressed as percentage of non-treated cells
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measured by image analysis as described in methods. Data represent mean  SEM values of 3
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independent experiments performed in quadruplicates. Significance: **: p<0.01 and #: p<0.001,
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was calculated using ANOVA test.
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Figure S4: Impact on signaling pathways of CXCR4 variants. (a) ERK1/2 phosphorylation
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status upon CXCL12 stimulation measured by flow cytometry and expressed as ratio of non-
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treated Kasumi-1 cells expressing empty vector. Data represent mean  SEM values of 3
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independent experiments performed in duplicates. Significance: *: p<0.05, was calculated using
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ANOVA test. (b) Calcium efflux in Kasumi-1 cells upon CXCL12 stimulation measured by
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spectrofluorimetry. Data represent mean  SEM values of 3 independent experiments performed
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in duplicates. Significance: #: p<0.001, was calculated using ANOVA test).
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Figure S5: Impact of CXCR4 variants expression on the phenotype of Kasumi-1 cells. (a)
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Infiltration of Kasumi-1 cell variants in the liver 7 days after transplantation (hCD34 stain). (b)
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Surface expression of CXCR7, CD34, CD49E/D and CD62L on Kasumi-1 cells expressing
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empty vector (Mock, open thin dotted black histogram), CXCR4-WT (open black histogram),
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CXCR4-S339A (open red histogram) or CXCR4-S339E (open green histogram) measured by
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flow cytometry. Isotype is depicted as filled light gray histograms, Jurkat cells were used as
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positive controls except for CXCR7 surface expression where MCF-7 cells were used as positive
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control (dark gray histograms). One representative experiment out of 3 is shown. (c) Apoptosis
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associated factors expression levels determined by immunoblots with antibodies against the
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indicated factors. One representative experiment out of 2 is shown.
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Figure S6: In vitro cell adhesion capacity on TNFα activated HUVECs and stroma
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associated resistance of CXCR4 variants expressing Kasumi-1 cells. (a) Adhesion capacity
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of CXCR4 variants expressing Kasumi-1 cells on HUVECs activated by TNFα (1 hour,
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20ng/ml). Expressed as relative units of GFP-positive cell density. Data represent means  SEM
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of 3 independent experiments performed in duplicates. Significance: **: p<0.01 and #: p<0.001,
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was calculated using ANOVA test. (b) Detachment of CXCR4 variants expressing Kasumi-1
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cells from HUVECs activated by TNFα (1 hour, 20ng/ml) and exposed to CXCL12 (50nM) for 30
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min. Expressed as ratio of GFP-positive cell compared to Mock cells, data represent means 
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SEM of 3 independent experiments performed in duplicates. Significance: **: p<0.01 and #:
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p<0.001, was calculated using ANOVA test. (c) Stromal MS-5 cells protect Kasumi-1 cells
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expressing different CXCR4 variants from Ara-C (5µM) induced apoptosis. Data represent mean
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 SEM values of 3 independent experiments performed in duplicates. Significance: *: p<0.05,
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was calculated using an unpaired t-test.
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