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Blood, Vol. 112, Issue 3, 721-732, August 1, 2008
Elevated protein tyrosine phosphatase activity provokes Eph/ephrin-facilitated adhesion of pre-B leukemia cells
Blood Wimmer-Kleikamp et al.
112: 721
Supplemental materials for: Wimmer-Kleikamp et al
Files in this Data Supplement:
- Figure S1. EphrinA5-induced cytoskeletal contraction requires active EphA3 kinase (JPG, 85.2 KB)
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AO2 melanoma cells with undetectable endogenous EphA39 were stably transfected with expression vectors encoding w/t or mutant EphA3 as indicated: cytoplasmic truncated (Y570stop), kinase-inactive (K653M) or tyrosine replaced (3YF) mutants. (A) Cells were stimulated with clustered ephrinA5 (+) or left untreated (-), and fixed cells stained with Alexa-labelled phalloidin for confocal microscopy. (B) Adherent AO2 cell clones, in comparison to EphA3-positive LiBr melanoma cells, before (blue bars) and after (yellow bars) stimulation with pre-clustered ephrinA5 were quantitated by XTT assay. The value in wells of non-stimulated cells was set at 100%, the mean and standard deviation of triplicate samples is shown.
- Figure S2. Expression of EphA3, but not of other Ephs or ephrins is highly elevated in LK63 cells (JPG, 86.7 KB)
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The mRNA and cell surface protein expression levels of various Eph receptors and ephrins were assessed by (A) Q-PCR and (B) flow cytometry. A number of Eph and ephrin proteins fused to the Fc portion of human IgG were used in combination with FITC-labelled anti¬human Fc antibodies to detect corresponding binding partners, as indicated (left panel): ephrin-A5 Fc binding predominantly A-type Eph receptors and EphB2, ephrin-B2 binding B-type Ephs, EphA3 Fc binding A-type ephrins, EphA4 Fc also binding ephrin-B2 and ephrin¬B3 and EphB2 binding all B-type ephrins.1 In addition, a number of mouse monoclonal antibodies against EphA1, EphA2, EphA3 (IIIA4 mAb), and EphA4 were used for analysis (middle panel). Cell surface expression of pre B-cell markers CD19 and VLA was assessed with monoclonal antibodies and corresponding FITC-labelled anti-mouse secondary antibodies. The M2 monoclonal antibody against the FLAG peptide was used as negative control as indicated.
- Figure S3. Association of FAK with EphA3 does not change during LK63 adhesion (JPG, 82.7 KB)
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(A) The distribution of EphA3 and of FAK was analysed in LK63 cells adhering to ephrin-A5 Fc-coated glass cover slips by optical sectioning on a confocal microscope. Alexa594 secondary antibodies (red pseudo-colour) were used to detect  -EphA3 antibodies, Alexa488 secondary antibodies (green pseudo-colour) to detect -FAK antibodies. Optical sections at the top, at the middle, and at the LK63 cell surface facing the glass slide (bottom) are illustrated. Scale bar: 10 µm. (B) LK63 cells were adhered onto FN- or ephrinA5-coated culture dishes for indicated times. FAK and EphA3 were immunoprecipitated from whole cell lysates and analysed in Western blots as indicated. The zero (0) time point illustrates suspended LK63 cells.
- Figure S4. Extracellular hydrogen peroxide promotes EphA3 phosphorylation in a dose-dependent manner (JPG, 74.8 KB)
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EphA3 was immunoprecipitated from Triton-X100 lysates of EphA3 transfected HEK 293 cells, AO2 melanoma cells, or LK63 leukaemia cells that had been treated with pervanadate or hydrogen peroxide at indicated concentrations. IP’s were analysed by -PY Western blots. The EphA3 levels in whole cell lysates were analysed in parallel to assess even loading.
- Figure S5. Association with EphA3 and effect on autophosphorylation by cytosolic PTP’s (JPG, 71.8 KB)
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(A) Association of endogenous PTP-PEST, LMW-PTP and SHP-2 with EphA3 in EphA3/HEK293T, LK63 leukaemia and LiBr melanoma cells. EphA3 IP’s of ephrinA5¬stimulated (+) and non-stimulated (-) cells were analysed by Western blot with antibodies against LMW-PTP, PTP-PEST and SHP-2, as indicated. (B) EphA3/HEK293T cells, transiently transfected with PTP-PEST –siRNA (left panel) or PTP-PEST –cDNA (right panel), were stimulated with pre-clustered ephrinA5-Fc (+) or left untreated (-). EphA3 PY levels were compared to those in respective control samples (cyclophyllin control siRNA or empty pMT2 vector) by Western blot analysis.
- Video 1. LK63 cells were added to an ephrinA5-Fc coated coverslip, placed inside a temperature (37°C) and CO2 (10%) -controlled climate chamber on a Leica AF6000LX Life Cell Imaging system and bright-field images taken every minute using a 63×, 1.3 NA glycerol lens (MOV, 807 KB)
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The first frame of SM1 illustrates a cell that just attached in the lower left corner of the section and extends large lamellopodia.
- Video 2. Cell in the centre of the section which rapidly extends and retracts large, uropod-like protrusions (MOV, 1.86 MB)
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Representative frames of movies SM1 and SM2 are shown in Fig. 1C.
- Video 3. Protein-A coated biobeads conjugated with Alexa546 ephrinA5-Fc were added to a mononlayer of EphA3 over-expressing HEK293T cells (MOV, 1.42 MB)
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Confocal fluorescence microscopic images were taken at 1 min intervals. Within 30 min fluorescent patches increasingly mark the cell perimeter, and within 45 min fluoresent vesicles are apparent within several cells. Towards the end of the time course, many cells in direct contact or in close proximity to the beads display distinctively fluorescent cell membranes and cytosolic vesicles. Representative frames of the movie, at 5, 15, 45 and 80 min time points are shown in Fig. 5A.
- Video 4. In a parallel experiment, LK63 cells were added to Alexa546 ephrinA5-Fc conjugated beads (MOV, 1.36 MB)
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Confocal fluorescence and brightfield microscopic images were taken at 1 min intervals and merged images are shown. The Alexa546 fluorescence in these merged images can be distinguished by red “pseudo-colouring.” While LK63 cells bind avidly to the ephrinA5-conjugated beads (attachment of a single LK63 cell to a bead is apparent at 28 – 35min), there is no evidence of ephrin cleavage or internalisation throughout the experiment. Representative frames of the movie, at 5 and 80 min time points, are shown in Fig. 5C.
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