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Fig. 4. EGF stimulates enhanced chemotaxis and reduced epithelial integrity in ß4(AD) cells dependent upon an alternate pathway utilizing RhoA and integrin {alpha}3ß1. (A) Effect of ß4(AD) expression upon monolayer scratch migration. Cells were prepared as in Fig. 1A and incubated without growth factors (top panel) or with 2 ng/ml EGF (lower panel) for 24 hours at 37°C. Scale bar: 300 µm. (B) Effect of ß4(AD) expression upon transwell chemotaxis. Transwell experiments with ß4(AD) keratinocytes were performed with collagen IV-coated transwells and 2 ng/ml EGF. Cells were incubated for 16 hours with media supplemented in upper and lower chambers with IgG1, inhibitory ß4 integrin antibody ASC-8, inhibitory {alpha}3 integrin antibody P1B5 or inhibitory laminin-5 antibody BM165. Actual induction IgG 60.0±4.2 cells/field, ASC-8 60.0±4.4 cells/field, P1B5 10.0±1.2 cells/field, BM165 16.1±1.1 cells/field. (C) Effect of GTPase inhibition upon ß4(AD)-dependent chemotaxis. ß4(AD) cells were retrovirally transduced with control LacZ or inhibitory GTPase constructs, N17Cdc42, N17Rac1 or N19RhoA. Chemotaxis experiments were performed with collagen IV-coated transwells and 2 ng/ml EGF. Actual induction, ß4(AD)LacZ 136±23.1 cells/field, ß4(AD)N17Rac1 15.0±3.7 cells/field, ß4(AD)N19RhoA 21.0±3.6 cells/field, ß4(AD)N17Cdc42 8.4±2.7 cells/field. (D) Effect of ß4(AD) expression upon cell scattering. Cells were incubated at low density for 4 days in SFM before being photographed under phase contrast illumination. Scale bar: 40 µm. (E) Effect of EGF upon cell scattering. Cells were grown in growth factor free SFM for 4 days and scattered colonies counted using criteria described in materials and methods (light shaded columns). Cells were then incubated for 16 hours with 2 ng/ml EGF and colony scatter counts repeated (dark shaded columns). For antibody treatments, ß4(+) cells were incubated for 3 days in normal SFM then 1 day in growth factor free SFM with 10 µg/ml mouse IgG or ß4 integrin inhibitor ASC-8, before colony counts (light shaded columns). Colony scattering was measured after 6 hours with 2 ng/ml EGF (dark shaded columns). Each point represents the data from at least 50 colonies (n=3). (F) Effect of N19RhoA expression upon ß4(+) transwell chemotaxis. Transwell experiments with ß4(+)LacZ and ß4(+)N19RhoA keratinocytes were performed with fibronectin-coated transwells and 2 ng/ml EGF. Cells were incubated for 16 hours with media supplemented in upper and lower chambers with IgG1 or inhibitory ß4 integrin antibody ASC-8. Actual induction, ß4(+)LacZ IgG 116.5±18.5 cells/field, ß4(+)LacZ ASC-8 113.5±16.9 cells/field, ß4(+)N19RhoA IgG 77.7±7.5 cells/field, ß4(+)N19RhoA ASC-8 34±21.3 cells/field.