FIG. 6.

Decreased migration and invasion of GBM10 cells exposed to TMZ and nutlin3a compared with single-agent exposure. A: GBM10 cells were treated for 8 hours with vehicle (V), 25 μM TMZ (T), 10 μM nutlin3a (N), or T/N; p53, MDM2, and GAPDH expression were analyzed by Western blot. B: A monolayer of GBM10 cells was wounded to generate a cell-free gap as described in the Methods. The cell-free gap was photographed to record the wound width (red arrows) at Hour 0. GBM10 cells were then treated for 12 hours with vehicle, 25 μM TMZ, 10 μM nutlin3a, or T/N. Cells were observed microscopically every 2 hours to monitor closing of the gap, and photographs were taken again at the marked wound location for measurement of migration. Representative photographs at 12 hours are shown. Each experiment was repeated 3 times and data are the mean ± SD of the 3 repetitions. Post hoc analyses demonstrated that all treatment pairwise comparisons were significant (*** = p < 0.001), except vehicle versus TMZ and nutlin3a versus T/N. C: GBM10 cells were incubated in fibronectin-coated insert wells and exposed to TMZ and nutlin3a as described above. After 8 hours of incubation, the filters were removed and fixed with methanol for 15 minutes; the cells on the upper side of the membrane were removed. Invasive cells, which were able to breach 8-μm pores and grow on the lower side, were stained with 1% crystal violet. Representative photographs at 8 hours posttreatment are shown. The cells that had migrated to the lower side of the membrane were counted under an inverted microscope in 5 fields selected randomly (100× magnification). Each experiment was repeated 3 times. Post hoc analyses demonstrated that all treatment pairwise comparisons were significant (** = p < 0.01, *** = p < 0.001), except vehicle versus TMZ.