Figure 3

HB-EGF and OSM promote epithelial cell migration. (A). HB-EGF secreted by primed MΦ induces chemotaxis of SCC-9 cells. MΦ were cultured for 6 days in either control medium or 50% MDA-MB-231-conditioned medium. Subsequently, cells received 50% MDA-MB-231-conditioned serum-free medium or RPMI for 24 hrs. MΦ supernatants were collected and used as chemoattractants for SCC-9 cells in Boyden chamber assays. Where indicated, media were preincubated with control Ig (C), an HB-EGF blocking antibody (H) or 250 nM EGFR kinase inhibitor AG1478 (AG). Representative experiment with corresponding micrographs. N: culture medium; P: priming medium; MΦ: supernatant of control macrophages; P-MΦ: supernatant of primed macrophages; scale bar: 200 μm; n = 3; *p < 0.05 by Student’s T-Test. (B). OSM promotes motility of MCF7 cells. Cells were incubated for 5 days with or without the indicated concentrations of recombinant OSM. Subsequently, cells were plated into Boyden chambers, using the same dose of OSM in RPMI + 2% FCS in the upper and lower compartments. (C). OSM promotes motility of SCC-9 cells. Cells were incubated for 48 hrs with or without 10 ng/ml recombinant OSM, and were subsequently plated into Boyden chambers, using the same dose of OSM in RPMI in the upper and lower compartments. Results are means ± SEM; n = 4; *p < 0.05 by Student’s T-Test. (D). HB-EGF and OSM cooperate in promoting chemotaxis of MCF 10A cells. Cells were cultured for 48 hrs with or without the indicated concentrations of recombinant OSM. Subsequently, cells were plated into Boyden chambers, using the same concentration of OSM in RPMI in the upper and lower compartments. Where indicated, 2 ng/ml recombinant HB-EGF was used as a chemoattractant in the lower compartment. Results are means ± SEM; n = 3; *p < 0.05 by Student’s T-Test; micrographs show a representative experiment; scale bar: 200 μm.