Figure 7.
Quantitative evaluation of the effects of OSU8-1 and EGFR inactivation on keratinocyte growth and migration. (a) Effect of 1 μM OSU8-1 on juxtacrine activity of EGFR ligands in cultured keratinocytes. Juxtacrine activity was measured as described in Materials and Methods. (b) The effect of OSU8-1, antibody No. 425 and AG1478 on the growth of keratinocytes. Keratinocytes were seeded at a density of 105 cells/well in 6-well plates and incubated for 3 d in the absence or presence of OSU8-1 (1 μM), antibody No. 425 (10 μg/ml), or AG1478 (30 nM). (a and b) Values represent the average of triplicate experiments. (c) Stimulation of keratinocyte migration by HB-EGF. The ability of recombinant HB-EGF to stimulate keratinocyte migration was tested using a Boyden chamber. (d) Stimulation of keratinocyte migration by conditioned medium. Serially diluted, 48-h conditioned medium obtained from wounded keratinocytes was added to the bottom wells of Boyden chambers and tested for its ability to stimulate keratinocyte migration. Open circles, conditioned medium generated in the presence of 1 μM OSU8-1; closed circles, conditioned medium obtained without 1 μM OSU8-1. (e) Effect of OSU8-1 on the migration response of keratinocytes induced by conditioned medium from wounded keratinocyte cultures. The indicated concentrations of OSU8-1 were added to the top (open circles) and the bottom (closed circles) wells of a Boyden chamber containing wounded keratinocyte conditioned medium in the bottom wells. An open triangle shows migrated cell number obtained using fresh medium in the bottom wells as a control. (c–e) Each point is the average of quadruplicate measurements.