Blood, Vol. 112, Issue 1, 100-110, July 1, 2008

Nuclear translocation of urokinase-type plasminogen activator
Blood Stepanova et al. 112: 100

Supplemental materials for: Stepanova et al

Surface plasmon resonance analysis
The interaction between WT-scuPA and soluble uPAR or recombinant nucleoilin was analyzed on a model 3000 Biosensor (Biacore, AB). uPAR and nucleolin were covalently immobilized to the surface of sensor chip CM5 (research grade) by amine coupling in 10 mM acetic buffer, pH 5.5 and pH 4.5, respectively. The cell activated and blocked in the absence of the ligand, and cell coated with Fab fragments of polyclonal mouse antibodies were used as the negative controls. scuPA was transferred to DPBS-T buffer (DPBS (Invitrogen) containing 0.2% Tween-20 (Sigma) at pH 5.5, 6.0, 6.5 or 7.4) using Micro BioSpin 6 columns (Bio-Rad). Samples were injected into the flow cells at 12 analyte concentrations ranging from 0.25 to 600 nM at a flow rate of 2 µl/min at 25°C at association and dissociation times of 2 hr and 10 min, respectively. Injection mode KINJECT, optimized for kinetic measurements, was used in all experiments. To regenerate the surface, 0.1 M glycine-HCl, pH 3.0, was applied for 5 min at a flow rate of 2 µl/min. The signal from the corresponding control surface was subtracted and the Kd based on steady state affinity mode was calculated using BIAevaluation version 4.1 software as described⁴³.

Solid phase binding assay
Mouse recombinant FLAG-nucleolin (5 µg/ml) was immobilized overnight at 4°C in DPBS on ELISA strip-well plate (Corning-Costar). Control wells were coated with BSA (3 mg/ml) for 18h at 4°C. All wells were blocked with 2% BSA in PBS (pH 7.4) for 1 h. ¹²⁵I-scuPA (12-400 nM) was added in PBS/0.2%Tween-20/2% BSA with indicated pH (5.5-7.4), incubated for 1h and washed. Bound radioactivity was measured by counting of individual wells in a -counter. Radioactivity values obtained in BSA-coated wells were subtracted from nucleolin-associated radioactivity values to obtain specific binding numbers. All assays were performed in triplicates.

Files in this Data Supplement:

• Figure S1. ΔGFD-scuPA translocates to the nucleus in HeLa cells (JPG, 91.6 KB) -
  HeLa were incubated for 30 min with 10 nM GFD-scuPA in complete medium at 37°C, washed and fixed in cold MeOH for 15 min. To visualize uPA, the fixed cells were incubated with rabbit -uPA pAb and Alexa488-conjugated goat -rabbit pAb. Nuclei were stained with DAPI. Images were taken using a Zeiss LSM 510 laser scanning confocal microscope with the z interval set at 0.3 µm. Upper panels: Confocal images taken through a single 0.3 µm optical slice. Left: Staining with -uPA Ab is shown in green. Middle: Counterstain of the nucleus with DAPI is shown as pseudo-color in red. Right: merged images within one focal plane. Lower panels: Sequences of merged images taken in various z sections at 0.3 mm intervals were subjected to 3-dimensional reconstitution using “Volocity 4.1.0” software (Improvision Inc). Reconstitution of virtual 3D images taken from the cell sectioned along the XY, XZ and YZ planes as shown in Panel a. Panel b: Sectioning along the XY plane (red-lined box). Panel c: Sectioning along the YZ plane (blue-lined box). Panel d: Sectioning along the XZ plane (green-lined box). Analysis of the 3D sectional images reveals a substantial fraction of scuPA and GFD-scuPA within the nucleus.
  
  
  
  
  
  
• Figure S2. Immunodetection of uPA in untreated cells (JPG, 41.1 KB) -
  BJ cells (upper panels) and HeLa cells (bottom panels) were incubated in complete medium, washed and fixed in cold MeOH for 15 min. The fixed cells were stained with rabbit -uPA pAb and Alexa488-conjugated goat -rabbit pAb. Nuclei were visualized with DAPI. Images were taken using a Zeiss LSM 510 laser scanning confocal microscope with the laser scanning parameters same as for Figure 1 in the manuscript.
  
  
  
  
  
  
• Figure S3. ¹²⁵I-WT-scuPA associates with immobilized nucleolin in a solid phase binding assay (JPG, 42.2 KB) -
  Mouse recombinant FLAG-nucleolin (5 µg/ml) was immobilized on ELISA strip-well plate, blocked with 2% BSA in PBS/0.2% Tween-20 (pH 7.4). Control wells were coated with BSA (3 mg/ml). ¹²⁵I-WT-scuPA (200 nM) was added in PBS/0.2%Tween-20/2%BSA with indicated pH, incubated for 1h at RT and washed. Bound radioactivity was evaluated by counting of individual well in -counter. Radioactivity values obtained in BSA-coated well were subtracted from nucleolin -associated radioactivity values to obtain specific binding numbers. All assays were performed in triplicates. Inset: Dose-dependence of ¹²⁵I-WT-scuPA binding to immobilized nucleolin at pH 7.4.
  
  
  
  
  
  
• Figure S4. Immunodetection of ΔK-scuPA in HeLa cells (JPG, 31.4 KB) -
  HeLa cells were incubated for 30 min with 10 nM K-scuPA in complete medium at 37°C, washed and fixed in cold MeOH for 15 min. The cells were stained and observed as in Fig. 1S. Confocal images were taken through a single 0.3 µm optical slice. Left: Staining with -uPA Ab is shown in green. Middle: Counterstain of the nucleus with DAPI is shown as pseudo-color in red. Right: merged images within one focal plane.
  
  
  
  
  
  
• Figure S5. S³⁵⁶A-scuPA does not induce α-SMA expression in BJ cells (JPG, 62.5 KB) -
  (A) Subcellular distribution of ¹²⁵I-S³⁵⁶A-scuPA in BJ cells. BJ cells were incubated with 10 nM ¹²⁵I-S³⁵⁶A-scuPA for 1 hr at 37°C, washed and the subcellular fractions were isolated as in Fig. 2. The radioactivity in each fraction was measured and normalized per 10⁶ cells to compare the absolute amounts of proteins incorporated into each fraction. The results of one experiment, representative of three so performed, are shown. (B) S³⁵⁶A-scuPA effect on -SMA expression in BJ cells. BJ cells were serum-starved for 24 hr and serum-free medium containing 1-10 nM S³⁵⁶A-scuPA was added for 24 hr. Cell lysates were prepared as in “Methods,” analyzed by SDS-PAGE and WB using mouse anti--SMA MAb and -GAPDH MAb. The results shown are from one experiment representative of 3 so performed.
  
  
  
  
  
  
• Video 1. HeLa cells were treated and stained as described in Fig. 1A. scuPA is stained green and the nucleus outlined by DAPI staining is shown as pseudo-color in red (AVI, 17.7 MB) -
  Images were taken using a Zeiss LSM 510 laser scanning confocal microscope with the z interval set at 0.3 µm. Sequences of merged images taken in various z sections at 0.3 µm intervals were subjected to 3-dimensional reconstitution using “Volocity 4.1.0” software (Improvision Inc). The movie shows a sequence of 3D view snapshots taken at different view angles.
  
  
• Video 2. BJ cells were treated and stained as described in Fig. 1B (AVI, 15.8 MB) -
  Staining for GFD-scuPA is shown in green and the nucleus outlined by DAPI staining is shown as pseudo-color in red. The movie was created as described above.