FIGURE 5.

Purified suPAR activates ERK1/2 and promotes migration of GBM cells. A, purified recombinant uPAR (10 nm) was incubated with parental U373MG cells (parental) or with EGFRvIII-expressing U373MG cells, which had been serum-starved for 18 h, for the indicated time points. Immunoblot analysis was performed to detect phospho-ERK1/2, total ERK1/2, phospho-Akt, and total Akt. Mean relative signal intensities for P-ERK1/2 and P-Akt relative to T-ERK1/2 and T-Akt are shown below the blots (n = 3). B, parental U373MG cells were serum-starved for 18 h, added to Transwells, and allowed to migrate toward 1% serum supplemented with 10 nm suPAR or vehicle (Ctrl) for 18 h. The number of cells that migrated to the underside of the membrane were counted and expressed relative to the control (mean ± S.E.; n = 3, ***, p < 0.001). C, cell migration was studied as in B with parental and EGFRvIII-expressing U373MG cells. The concentration of suPAR was varied as shown (mean ± S.E.; n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001). D, parental U373MG cells were allowed to migrate in Transwells for 18 h. The bottom chamber contained serum-free medium and 15 nm suPAR (+) or vehicle (−), as indicated. Other additives to the lower chamber included uPAR-neutralizing antibody (α-uPAR, 50 μg/ml ATN-658 + 15 μg/ml MAB807) or control mouse IgG (65 μg/ml). The number of cells that migrated to the underside of the membranes are expressed relative to the control, which was not suPAR-treated (mean ± S.E.; n = 3; **, p < 0.01). E, parental U373MG cells were allowed to migrate toward 1% serum-containing medium and 15 nm suPAR for 18 h, as indicated (+). 20 μm PD98059 was added to both chambers, as indicated (+). The number of migrated cells were determined and expressed relative to the vehicle-treated control (mean ± S.E.; n = 3; **, p < 0.01; *, p < 0.05). F, qPCR was performed to detect uPA mRNA after treating parental U373MG cells with suPAR for 4 h (mean ± S.E.; n = 3).