Effect of MAPK/Erk and PI3K/Akt pathway inhibitors on HER1 ligand secretion. HMEC lines expressing different levels of HER receptors were preincubated with a single inhibitor (20 μg/ml LY 294002, 200 nM wortmannin, 25 µM PD 98059 or 10 µM U0126) for 1 h, followed by treatment with 12 ng/ml EGF for 8 h. DMSO concentration was 0.1% in all experiments except those marked as "ctrl". Conditioned medium (CM) was collected and the concentrations of A) AREG, B) HB-EGF and C) TGF-α were determined by ELISA microarray analysis. Ligand concentrations were normalized with respect to the concentration in the HER1 cells treated only with 12 ng/ml EGF for 8 h. Columns and crossbars represent the mean and standard deviation, respectively, from five replicates, each of which was from a separate culture dish. The standard deviation is shown for every analysis, but in some cases the crossbar may be too small to be visible. The asterisks denote statistically significant differences between inhibitor-treated cells and ctrl cells for the individual cell lines, as determined by both analysis of variance and by Fisher's protected least significant difference.