FIGURE 1:

Inhibition of EphA2-dependent cell retraction by representative hit compounds. (A, B) “No compound” refers to cells treated with Fc control or ephrin-A1 Fc and DMSO. Other cells were pretreated for 40 min with the indicated compounds and then for 10 min with Fc or ephrin-A1 Fc in the presence of the compounds. Forskolin was used at 20 μM and dibutyryl cyclical AMP (dbcAMP) at 0.5 mM. The other compounds were used at 5 μM. NECA, 5′-(N-ethylcarboxamido) adenosine. Cells were stained for F-actin with fluorescent phalloidin (red), and nuclei were labeled with DAPI (blue). The inactive compound (O100, oxotremorine methiodide) did not inhibit retraction and rounding of the cells induced by ephrin-A1 Fc, similar to the control. All other compounds shown inhibited retraction, and the cells remained flat. (C) Dose–response curve for forskolin-mediated inhibition of cell retraction analyzed by automated image analysis in the 384-well format. Averages and SEs from quadruplicate measurements from two experiments carried out on different days.