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Blood, Vol. 112, Issue 1, 169-178, July 1, 2008
 4β1 integrin and 190-kDa CD44v constitute a cell surface docking complex for gelatinase B/MMP-9 in chronic leukemic but not in normal B cells
Blood Redondo-Muñoz et al.
112: 169
Supplemental materials for: Redondo-Muñoz et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 916 KB)
- Figure S1. Constitutive interaction of proMMP-9 with α4β1 integrin and 190 kDa CD44v in B-CLL cells (JPG, 53.6 KB)
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2 × 107 fresh B-CLL cells (which had not been in culture for 24 h) from two patients were lysed and immunoprecipitated with anti-MMP-9 pAbs or with rabbit IgG (Ctl). (A) An aliquot of the immunoprecipitates was analyzed by gelatin zymography on 10% gels and proMMP-9 was identified as a major 92 kDa form. (B) Western blotting analysis of the same lysates shown in (A) using the indicated specific Abs.
- Figure S2. Blocking the function of α4β1 integrin or CD44 reduces the levels of membrane-bound proMMP-9 (JPG, 73.5 KB)
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(A) 3 × 106 B-CLL were incubated in RPMI, 0.1% FCS in the absence (control) or presence of the indicated mAbs or peptides. After 24h cells were lysed and the membrane fraction and conditioned media anayzed by gelatin zymography. Values (arbitrary units) represent the average of the three patients studied after normalizing control values to 100. *p≤0.05, **p≤0.01. (B) B-CLL cells from two patients were incubated as described in (A) with or without anti-CD44 or anti-HLA mAbs. Membrane fractions and conditioned media were analyzed by gelatin zymography. *p≤0.05.
- Figure S3. Viability of B-CLL cells after transfection with siRNAs for β1 or CD44 (JPG, 85.4 KB)
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2 × 106 B-CLL cells from two patients were transfected with the indicated siRNAs. After 16 h ( 1 siRNA) or 24 h (CD44 and MMP-9 siRNA), the FCS concentration was reduced to 0.1% and cells incubated for an additional 24 h period. Cells were then analysed by flow cytometry using Annexin V-FITC and propidium iodide. Values indicate the percentage of viable cells for each condition.
- Figure S4. Western blot analysis of B-CLL cell lysates after transfection with β1 or CD44 siRNAs (JPG, 74.1 KB)
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B-CLL cells from 3 patients (western blots shown for two representative patients) were not transfected or transfected with control siRNA (Ctl) (A) (B), two different 1 siRNAs (A), or two different CD44 siRNAs (B). Cells were lysed and 1 and CD44 expression analyzed by western blotting. Quantitative values represent the 1/actin (A) or CD44/actin (B) ratio for all three patients studied, after normalizing 1 (A) and CD44 (B) levels in untransfected cells to 1. *p≤0.05, **p≤0.01, ***p≤0.001.
- Figure S5. Recovery of surface-bound proMMP-9 levels upon cell culture of siRNA-transfected B-CLL cells (JPG, 83.8 KB)
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(A) 2 × 106 B-CLL cells (two patients) were untransfected (None) or transfected with 1, CD44 or control siRNA as described. At the indicated times after the initial 16 h (1) or 24 h (CD44, control), cell membrane fractions were analyzed by gelatin zymography. Values represent the average of the two patients after normalizing control values to 100. (B) The same siRNA-transfected cells shown in (A) were analyzed by flow cytometry at the indicated times using anti-1, anti- 4 or anti-CD44 mAbs.
- Figure S6. Further characterization of the binding of soluble proMMP-9 to B-CLL cells (JPG, 66.9 KB)
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(A) 5 × 105 B-CLL cells from three patients were incubated with or without 10 µg/ml proMMP-9 for 30 min at 4°C, washed and maintained at 4°C. At the indicated times, anti-MMP-9 and secondary Abs were added and MMP-9 surface expression analyzed by flow cytometry. (B) 5 × 105 B-CLL cells were incubated at 4°C with the indicated concentrations of proMMP-9. After 30 min, MMP-9 surface expression was analyzed by flow cytometry as explained in (A). (C) B-CLL cells (two patients) were incubated (30 min, 4°C) with 10 µg/ml proMMP-9, washed and further incubated with anti-MMP-9, anti-1, or anti-transferrin receptor Abs. Cells were washed and placed at 4°C or 37°C. At the indicated times, surface expression of MMP-9 or transferrin receptor was analyzed by flow cytometry. *p≤0.05, **p≤0.01, ***p≤0.001.
- Figure S7. The α4 and β1 integrin subunits co-immunoprecipitate with CD44 in B-CLL cells (JPG, 40.1 KB)
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Lysates from 2 × 107 B-CLL cells from two patients were immunoprecipitated with anti-CD44 or anti-HLA (Ctl) mAbs and analyzed by western blotting using the indicated Abs. The molecular weight of the various immunoprecipitated proteins is indicated.
- Figure S8. Lack of CD44 v6 exon on B-CLL cells and absence of v exons on normal B cells (JPG, 63.1 KB)
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B-CLL cells from two different patients, BRO168 cells, and peripheral blood B lymphocytes (PB-BL), were analyzed by flow cytometry using mAbs recognizing a conserved CD44 region (anti-CD44) or the indicated variable CD44 exons. BLM melanoma cells were used as positive control for CD44 v exons expression.
- Figure S9. Effect of CD44 or α4β1 integrin gene silencing on MMP-9 B-CLL cell surface localization (JPG, 84.8 KB)
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B-CLL cells were transfected with the indicated siRNAs and added to glass coverslips coated with 5 µg/ml poly-lysine. After 1 h at 37°C, cells were fixed and proteins visualized by confocal microscopy, after staining with specific Abs followed by Texas-Red- or Alexa 488-labelled-secondary Abs. Merged images of MMP-9 with 4, 1 or CD44 are shown for all three siRNA, together with their corresponding dot-plot analyses. Images were acquired using a confocal scanning inverted AOBS/SP2 microscope (Leica Microsystems, Heidelberg, Germany) with a 63×/1.3 NA PL-APO glycerol immersion objective. Leica’s LCS 15.37 dye-separation software was used for colocalization studies; when necessary, Adobe Photoshop 7.0 was used for image processing. Bar, 4 µm.
- Figure S10. Functional effect of exogeneous proMMP-9 on B-CLL cell migration and viability (JPG, 75.8 KB)
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(A, B) 5 × 105 B-CLL cells were incubated for 30 min with or without the indicated concentrations of proMMP-9 and added to Transwell filters coated with Matrigel (A) or TNF--activated HUVEC (B). 150 ng/ml CXCL12 was added to the medium in the bottom chamber, except for the control. After 24 h, migrated cells were counted by flow cytometry. Values (average of the three patients, each with similar results) are expressed as the % of the total number of cells added. *p≤0.05, **p≤0.01. (C) B-CLL cells from two of the patients used in (A) were incubated with 10 µg/ml proMMP-9 and after 24h, analyzed by flow cytometry using Annexin V-FITC and propidium iodide. Values indicate the percentage of viable cells for each condition.
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