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. Author manuscript; available in PMC: 2016 Jan 30.
Published in final edited form as: Oncogene. 2014 Oct 27;34(31):4078–4088. doi: 10.1038/onc.2014.336

Figure 5.

Figure 5

Effects of EGFRvIII neutralization on migration of U373MG GBM cells. (a) U373MG cells that express EGFRvIII under the control of a Dox repressible promoter were treated with Dox or with vehicle for 4 days. uPAR-specific siRNA (siuPAR) or NTC siRNA were introduced for 12 h. The cells were re-equilibrated in serum-containing medium for 24 h (with or without Dox) and then added to Transwell chambers. Cell migration was allowed to occur in Transwell chambers for 18 h. Serum was present exclusively in the lower chambers. Representative photomicrographs show cells that migrated to the underside surface of the membranes. (b) Cells that migrated to the underside surfaces of the membranes were counted (mean ± SEM, n=3, *, p<0.05). (c) U373MG GBM cells were cultured in the presence of Dox or vehicle (without Dox) for 4 days. These cells and ESC cell lines were transfected with NTC siRNA (black bars) or uPA-specific siRNA (grey bars). After re-equilibration in serum-containing medium for 24 h, cells were added to Transwell chambers. Cells were allowed to migrate for 18 h. The number of migrating cells was standardized against that observed with U373MG cells that were not treated with Dox and transfected with NTC siRNA (mean ± SEM, n=3, *, p<0.05; **, p<0.01). (d) EGFRvIII-expressing U373MG cells were treated with Gefitinib (1 µM) or vehicle (DMSO) for 4 days. The cells were transferred to SFM for 24 h. uPA mRNA expression was determined by qPCR and standardized against the level present in cells treated with vehicle (mean ± SEM; n=3, *, p<0.05). (e) EGFRvIII-expressing U373MG cells were treated with Gefitinib (1 µM) or vehicle (DMSO) for 4 days. Cell migration was allowed to occur in Transwell chambers for 18 h. The number of migrating cells was standardized against that observed with vehicle-treated U373MG cells (mean ± SEM, n=3, *, p<0.05). (f) EGFRvIII-expressing U373MG cells were treated with Gefitinib (1 µM) or with vehicle for 4 days and then transfected with uPA-specific siRNA (grey bar) or NTC siRNA (black bar). The cells were allowed to recover in serum-containing medium for 24 h (with continued treatment with Gefitinib or vehicle) before addition to Transwell chambers. Cell migration occurred for 18 h. The number of migrating cells was standardized against that observed with U373MG cells that were treated with vehicle and transfected with NTC siRNA (mean ± SEM, n=3, *, p<0.05).