Fig. 4

PDGF-AB enhances migration of BM-MSC through control of uPAR and β1-integrin association. a uPAR (green) and β1-integrin subunit (red) repartition in migrating cells. Images were acquired with a confocal microscope. uPAR and β1-integrin stainings were merged to show colocalization (yellow). Nuclei were stained with DAPI. Scale bars: 10 μm. b uPAR-β1 subunit coimmunoprecipitation. Two hours after adherence (Adh) on type I dermal collagen, cells were grown in serum-free control medium (−) or treated with PDGF-AB (+) for 1–18 hours. β1 integrin was immunoprecipitated from cell lysates with anti-β1 monoclonal antibodies or isotypic control IgG₁. Immunoprecipitates were analyzed by SDS-PAGE and blotted for β1-integrin subunit (top western blot) and uPAR (bottom western blot). (Left) Western blot representative of three experiments. T0, lysate of cells before seeding; Adh., lysate of cells allowed to adhere for 2 hours on type I dermal collagen. (Right) uPAR blots quantification by densitometry analysis using ImageJ software. Data expressed as the uPAR fold change after PDGF-AB treatment compared with serum-free control medium from three independent experiments (one donor per experiment). c Scratch test assay was performed on BM-MSC isolated from three different donors and seeded in plates coated with type I dermal collagen. Two hours after adherence, cells were grown in serum-free control medium or treated with PDGF-AB, supplemented or not with integrin blocking peptide β1P1 along with the corresponding scrambled control scβ1P1 or anti-uPAR neutralizing antibody. Results are expressed as percentages of wound closure at T = 22 h compared with T = 0. Mean of three independent experiments ± SEM are represented (one donor per experiment), each performed in triplicate. *P <0.05. PDGF-AB platelet-derived growth factor AB, uPAR urokinase-type plasminogen activator receptor