Figure 1.
Expression of murine Plaur in AT84 cells.
In vitro characterization of AT84 cells stably transfected with either empty vector (EV) or a vector containing cDNA encoding murine uPAR (Plaur). A: Western blot analysis of whole cell lysates using a polyclonal anti-murine uPAR antibody (AF534). A total of 7.5 ng of recombinant murine uPAR (rmuPAR) was loaded as a positive control. Re-probing for β-actin was used as a loading control. B: Western blot analysis of cellular membrane fractions using a polyclonal anti-murine uPAR antibody (AF534). Total protein was measured per sample and 53.5 µg of protein was loaded per lane. A and B: Images were cropped, as no additional bands were detectable. C: FACS analysis of non-permeabilized cells using a polyclonal anti-murine uPAR antibody (AF534). Alexa Fluor 488 anti-goat secondary antibody (A11055) was used as the secondary antibody. The quantified mean Alexa 488 fluorescence signal per cell line is presented in the panel to the right. D and E: Relative Plaur mRNA (uPAR) (D) or Plau mRNA (uPA) (E) expression levels as analysed using RT-qPCR. All expression levels were normalized to the expression of the reference genes Trfc and β-actin. Error bars represent the standard error of mean (+SEM) and N = 3. One-way ANOVA; *p<0.05. F: Plasminogen-gelatin (upper panel) and gelatin (lower panel) zymography analysis of conditioned medium of cells cultured for 24 hours in SFM. HMW-uPA and mPLM (mouse plasmin) were loaded as positive controls. The images were cropped to size. G: Relative Plasminogen mRNA (Plg) expression levels as analysed using RT-qPCR. Error bars represent standard error of mean (+SEM) and N = 3.
Figure 2.
Tumour microenvironment induced uPAR protein expression in tongue tumours.
Tumour growth pattern and uPAR protein levels in tongue tumours generated from the EV1, EV2, uPAR1 and uPAR2 cells. A–B: Representative images depicting the tumour growth pattern at the tumour-stroma interface in hematoxylin/eosin stained EV1 (A) and uPAR1 (B) tumours. Images were recorded at 10x magnification. C–D: Representative images depicting the IHC uPAR staining of the EV1 (C) or uPAR1 tumours (D). Images were recorded at 4x magnification. E–H: The images show high power magnification (20x magnifications) of the EV1 (E), uPAR1 (F), EV2 (G) and uPAR2 (H) tumours IHC stained for uPAR protein. Positive uPAR staining is seen as brown colour, and counterstaining was done with haematoxylin. I: The average staining index (SI) of the uPAR staining in the tumours. Maximum obtainable score is 9. The error bars shows the +SEM. N = number of tumours; EV1, N = 8/10; EV2, N = 5/10; uPAR1, N = 4/10; uPAR2 N = 9/10. One-way ANOVA; **p<0.01, *p<0.05. T = Tumours, S = Stroma.
Figure 3.
Knock-down of uPAR expression in AT84 cells.
shRNA knock-down of Plaur in AT84 cells. A: Flow chart showing the generation of the single cell clones. B: Western blot analysis of whole cell lysates from the single cell clones stably transfected with either shRNA-constructs (shRNA 3 = sh3, shRNA 4 = sh4 or shRNA 5 = sh5) targeting Plaur or constructs containing non-target shRNA (NT) or the empty vector (EV). uPAR was detected using a polyclonal anti-murine uPAR antibody (AF534). Re-probing for β-actin was used as a loading control. Images were cropped, as no additional bands were detected in the blot.
Figure 4.
In vivo tongue tumours of EV1 and uPAR1 knock-down cells.
IHC uPAR staining and growth pattern of tongue tumours generated from the EV1 and uPAR1 cells containing either shRNA targeting uPAR (EV1-sh and uPAR1-sh), or non-targeting shRNA (uPAR1-NT). A–C: IHC uPAR staining of EV1-sh (A), uPAR1-NT (B) and uPAR1-sh (C) tumours, respectively. Images were recorded at 20x magnifications. D: Representative image depicting the tumour growth pattern at the tumour-stroma interface in hematoxylin/eosin stained EV1-sh. E: The average SI of the uPAR staining in the tumours, with the maximum obtainable score of 9. The error bars shows the +SEM. N = number of tumours; EV1, N = 8/10; EV1-sh, N = 11/16; uPAR1, N = 4/10; uPAR1-NT, N = 3/8; uPAR1-sh, N = 4/16. One-way ANOVA; **p<0.01, *p<0.05. T = Tumour, S = Stroma.
Figure 5.
Leiomyoma stroma is a strong inducer of uPAR expression.
Representative images of low- (EV1-sh3) and high- (uPAR1-NT) uPAR-expressing cells invading the ex vivo leiomyoma tissue. Cells were incubated for 7 and 14 days, as indicated. The tissue was IHC stained for uPAR. Positive uPAR staining is seen as brown colour, counterstained with haematoxylin. Images were recorded at 10x magnification.
Figure 6.
Leiomyoma conditioned medium induced uPAR expression.
Analysis of uPAR expression induced by the LCM or purified ECM proteins in cultured uPAR knock-down cells. All Western blots were performed on whole cell lysates, and uPAR was detected using the polyclonal anti-murine uPAR antibody (AF534). A: Cells were either cultured in LCM (LM) or serum free medium (SF) for 24 hours. Cells were harvested with sample buffer and re-probing for β-actin was used as a loading control. B: uPAR1-NT cells were seeded on different ECM protein substrates, incubated for 24 hours and harvested using RIPA buffer. 7.5 µg of total protein was loaded per lane. Equal loading was verified by re-probing for β-actin. The poloxamer pluronic was used as a no-adhesion control. Col I = Collagen I, Vn = Vitronectin, Fn = Fibronectin, Lm = Laminin, ECL = Entactin, Collagen, Laminin, and FBS = Foetal Bovine Serum. C: Cells cultured in LCM (LM) or serum free medium (SF) for 24 hours were harvested using sample buffer and deglycosylated by PNGase F treatment (+) as indicated. Re-probing for β-actin was used as a loading control. The three bands detected by the anti-uPAR antibody are labelled 1, 2, and 3, respectively.
Figure 7.
Gelatinolytic activity is enhanced in tumours expressing high levels of uPAR.
ZBF-fixed tongue tumours were sectioned and analysed for the presence of gelatinolytic activity using DQ-gelatin in situ zymography. Gelatinolytic activity is seen as green fluorescence. A: Representative confocal images of tongue tumours generated from the uPAR1-NT cells (left panel) and EV1-sh3 cells (right panel). B: Quantification of fluorescence intensity (analysed using Volocity as described in materials and methods) for a minimum of 5 images per tumour, presented as mean values. Three individual uPAR1-NT tumours (No.1–No.3) and three EV1-sh tumours (No.1–No.3) were analysed. Error bars shows the standard deviation (+SD) between the five images analysed. Dark grey bars represents gelatinolytic activity in the tumour sections, light grey bars represents gelatinolytic activity in tumour sections treated with the metalloproteinase inhibitor EDTA. Mann-Whitney rank sum test; ***p<0.001, **p<0.01, *p<0.05.
Figure 8.
Gelatinolytic activity is enhanced in cells invading leiomyoma tissue.
ZBF-fixed leiomyoma tissue was sectioned and analysed for the presence of gelatinolytic activity using DQ-gelatin in situ zymography. Gelatinolytic activity is seen as green fluorescence, nuclei are stained blue with DAPI. Representative confocal images of cells expressing either low- (EV1-sh3, left panels) or high (uPAR1-NT, right panels) levels of uPAR invading the ex vivo leiomyoma tissue. The upper panels show gelatinolytic activity in the tissue, while the lower panels show tissue sections treated with the metalloproteinase inhibitor EDTA.