Differential effects of EREG on morphology, growth and migration of U87Ctrl and U87dn cells. (a) Morphological changes are selectively induced by EREG on U87dn cells. Cells were grown in the presence of 1% FCS with or without 30 ng/ml EREG. Photomicrographs of U87Ctrl and U87dn cells are shown after 3 days in culture. Bar = 50 μm. (b) Effects of EREG on U87 cell proliferation and migration. In the proliferation assay, cells were grown for four days. The total cell number was reported as fold-increase of the standard value (1.00) obtained with U87Ctrl cells in the absence of EREG. Results are the mean of triplicates ± SD. Mann–Whitney was performed for significance (*; p < 0.05). In the Transwell migration assay, cells were deposited in the migration chamber for 15 h and were then allowed to migrate for 9 h in the absence of serum, with or without EREG. Results were expressed as fold increase ± SD of the number of migrating cells in the presence vs. absence of EREG (*; p < 0.05). (c) EGF receptors are expressed in U87Ctrl and U87dn cells. Differential expression of ErbB1-4 mRNAs in U87dn versus U87Ctrl cells as depicted by transcriptomic (GEO, GSE22385; AffyID probe set numbers are indicated) and qPCR analyses. (d) Presence of EGFR (ErbB1) and ErB2 proteins in U87Ctrl and U87dn cells. For EGFR detection, cells were pre-incubated for 3 h in the absence of serum and were then stimulated or not with 30 ng/ml EREG for 20 min. Immunoblotting was performed using antibodies against EGFR, phospho-Tyr1173-EGFR (p-EGFR), ErbB2 or β-actin. Signal intensities of p-EGFR bands were quantified and normalized (N) to β-actin. The 1.0 value is used as the reference.