Fig. 1

uPAR cleavage is mediated by plasmin and uPA. AT84 cells stably transfected with either empty vector (EV)(AT84-EV) or a vector containing cDNA encoding mouse uPAR (AT84-uPAR) were analysed for uPAR mRNA and protein levels, secreted plasminogen activators and uPAR cleavage by Western blotting of whole cell lysates (A, F-I), flow cytometry (B), RT-qPCR (C) and plasminogen-gelatin zymography (plgzym)(D). a. AT84-uPAR (uPAR) or AT84-EV (EV) cells cultured in either serum-free medium (SFM) or medium containing 10% foetal bovine serum (FBSM) for 24- and 48 h. b. Non-permeabilized AT84-EV (pink: median fluorescence 371) and AT84-uPAR (purple: median fluorescence 853) cells. Negative control with no primary antibody added (filled curve). c. Relative uPAR mRNA (left panel) or uPA mRNA (right panel) expression levels. Error bars represent the standard deviation (+SD) and N = 3. Student T-test; * p < 0.05. d. Conditioned medium from cells cultured for 24- and 48 h in SFM. Positive control: mPLM (mouse plasmin). Active mouse plasmin (arrow), auto-proteolytic fragment of plasmin (black arrowhead), HMW-uPA (white arrowhead), and an unknown plasminogen activator (asterisk). e. Images of AT84-EV and AT84-uPAR cells in culture 24 h after seeding (10× magnification). f-i. Whole cell lysates of AT84-uPAR cells treated with PNGase F (+) or no PNGase F (−). f. Cells cultured in either FBSM or SFM. Glycosylated uPAR (uPAR_glc), deglycosylated samples gave rise to full length uPAR (uPAR_I-III) and cleaved uPAR (uPAR_II-III). g. Cells cultured for 24 h in FBSM (0), or FBSM supplemented with either 1.5 μM, 8 μM or 15 μM aprotinin, or 10 μM BC11 hydrobromide. h. Cells cultured for 24 h either in SFM without plasminogen (0), or in SFM supplemented with 1 nM, 10 nM or 100 nM plasminogen. i: AT84-uPAR cells cultured for 24 h in FBSM supplemented with 1-, 5-, 10- or 50 nM rmPAI-1. Controls (0 nM) received no additives