American Society of Hematology

Figure 7.

$Figure 7. Analysis of cell-associated MMP-9 in B-CLL cells. (A) B-CLL cells (3 × 106) from a representative sample were incubated on FN-H89 for 24 hours. Cells were removed and membrane and cytosolic fractions separated and subjected to gelatin zymography. Identical aliquots from the same lysate were analyzed by Western blotting using anti-RhoGDI–specific Abs. (B) B-CLL cells in medium containing 80 nM PMA were added to glass coverslips coated with 5 μg/mL p-Lys, 10 μg/mL FN-H89, or 10 μg/mL VCAM-1. After 1 hour at 37°C, podosomes were analyzed by confocal microscopy after staining F-actin (red) with Alexa 568–phalloidin and vinculin (green) with specific primary Abs/FITC-labeled secondary Abs. Inserts are ×20 magnifications. Bar represents 4 μm. (C) B-CLL cells were added to FN-H89– or VCAM-coated glass coverslips, and after 1 hour MMP-9 (green) was visualized with specific primary Abs and Alexa 488–labeled secondary Abs. F-actin (red) was stained as explained in panel B, and the merged images are shown. Colocalization (yellow) of MMP-9 and F-actin in podosomes was further demonstrated using dot-plot analyses as explained in “Materials and methods.” (D) B-CLL cells were preincubated or not for 1 hour with 30 nM wortmannin (Wmn) or 10 μM UO126 and added to glass coverslips coated with 10 μg/mL FN-H89. F-actin and MMP-9 were visualized as explained, and the merged images (yellow) and dot-plot analyses are shown. (E) B-CLL cells were added to coverslips coated with gelatin/fibronectin; after 24 hours, colocalization of actin and MMP-9 in podosomes was analyzed as explained. (F) B-CLL cells, with or without the indicated inhibitors, were added to coverslips coated with FITC-gelatin/fibronectin; after 24 hours, F-actin was stained with Alexa 568–phalloidin and sites of matrix degradation were visualized by the loss of green fluorescence. Arrows indicate cells containing podosomes. Bar represents 12 μm. 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 (Adobe Systems, San Jose, CA) was used for image processing.$

Analysis of cell-associated MMP-9 in B-CLL cells. (A) B-CLL cells (3 × 10⁶) from a representative sample were incubated on FN-H89 for 24 hours. Cells were removed and membrane and cytosolic fractions separated and subjected to gelatin zymography. Identical aliquots from the same lysate were analyzed by Western blotting using anti-RhoGDI–specific Abs. (B) B-CLL cells in medium containing 80 nM PMA were added to glass coverslips coated with 5 μg/mL p-Lys, 10 μg/mL FN-H89, or 10 μg/mL VCAM-1. After 1 hour at 37°C, podosomes were analyzed by confocal microscopy after staining F-actin (red) with Alexa 568–phalloidin and vinculin (green) with specific primary Abs/FITC-labeled secondary Abs. Inserts are ×20 magnifications. Bar represents 4 μm. (C) B-CLL cells were added to FN-H89– or VCAM-coated glass coverslips, and after 1 hour MMP-9 (green) was visualized with specific primary Abs and Alexa 488–labeled secondary Abs. F-actin (red) was stained as explained in panel B, and the merged images are shown. Colocalization (yellow) of MMP-9 and F-actin in podosomes was further demonstrated using dot-plot analyses as explained in “Materials and methods.” (D) B-CLL cells were preincubated or not for 1 hour with 30 nM wortmannin (Wmn) or 10 μM UO126 and added to glass coverslips coated with 10 μg/mL FN-H89. F-actin and MMP-9 were visualized as explained, and the merged images (yellow) and dot-plot analyses are shown. (E) B-CLL cells were added to coverslips coated with gelatin/fibronectin; after 24 hours, colocalization of actin and MMP-9 in podosomes was analyzed as explained. (F) B-CLL cells, with or without the indicated inhibitors, were added to coverslips coated with FITC-gelatin/fibronectin; after 24 hours, F-actin was stained with Alexa 568–phalloidin and sites of matrix degradation were visualized by the loss of green fluorescence. Arrows indicate cells containing podosomes. Bar represents 12 μm. 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 (Adobe Systems, San Jose, CA) was used for image processing.

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