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. 2012 Feb 15;23(4):533–542. doi: 10.1091/mbc.E11-01-0039

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© 2012 Ivkovic et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 1: — Effect of growth factor receptor stimulation and its inhibition on Transwell migration of C6 glioma cells. (A) C6 glioma cells expressing EGFR-mCherry (EGFR(+)) and untransfected C6 cells (EGFR(−)) were seeded into the upper part of a 3-μm-pore Transwell chamber. Migration across the Transwell membrane was measured after a 5-h incubation by staining the cells with 4,6-diamidino-2-phenylindole and counting the number of nuclei in 10 high-power fields. Data were normalized to the mean of EGFR(−) cells in the absence of EGF and expressed as fold enhancement. Bars represent mean ± 1 SD. EGFR(+) cells showed increased migration in response to EGF (10 ng/ml) compared with no EGF (p < 0.001). In comparison, EGFR(−) cells showed no appreciable migratory response to EGF (p = 1.00). (B) Transwell migration of EGFR(+) cells was monitored in the absence of serum or growth factor stimulation (Control), in the presence of 10 ng/ml EGF or 100 ng/ml PDGF, and in the presence of both (PDGF+EGF). Data were normalized to the mean of the control condition and expressed as fold enhancement. Bars represent mean ± 1 SD. Differences between EGF and PDGF conditions (p < 0.001) and between PDGF and EGF plus PDGF conditions (p < 0.005) were statistically significant. (C) Transwell migration in the presence of EGF, PDGF, and its cognate receptor inhibitors. While the EGFR tyrosine kinase inhibitor Iressa (10 μM) blocks Transwell migration in the presence of EGF, its effects are negated by addition of PDGF. Likewise, the PDGFR inhibitor Gleevec (20 μM) blocks PDGF-stimulated Transwell migration. Blebbistatin (10 μM) and Y27632 (50 μM) are also effective in the presence of EGF stimulation. Furthermore, unlike Iressa and Gleevec, blebbistatin also remains effective in the presence of combined EGF and PDGF stimulation. (D) Dose–response relationship for EGF-stimulated Transwell migration of EGFR(+) cells. Migration in response to EGF can be fit to a hyperbolic isotherm, defining an EC₅₀ of 0.28 ± 0.04 ng/ml (blue curve). In the presence of 10 μM blebbistatin (red curve), migration remains inhibited by > 90% over a range of EGF that spans two orders of magnitude.