Fig. 4.

The combined activation of ErbB2 plus TGFβ or Mek2 plus TGFβ leads to secretion of both EGFR-dependent and -independent soluble migratory factors. (A) MCF10A cells were seeded in transwell migration chambers, and conditioned media produced from cells as indicated was added to the bottom chambers and incubated for 18 h. *, P = 0.0056 as compared with TGFβ alone; P = 0.004 as compared with ErbB1 plus TGFβ. **, P = 0.008 as compared with TGFβ alone; P = 0.002 as compared with MEK2DD alone. (B) Migration assays were performed as described in A, except EGFR inhibitors AG1478 (300 nM) or mAb 225 (10 μg/ml) were added. Data are expressed as the percent of control cells that migrate or invade with inhibitor. Migration with Mek 2DD plus TGFβ-conditioned medium was not tested with mAb 225. (C) Migration assays were performed by using 10A.B2.TGFβ plus AP1510-conditioned medium and 5 μM U0126. Data are expressed as the percent of control cells that migrate with inhibitor. (D) Conditioned media from 10A.B2.TGFβ cells treated with AP1510 was made in the presence of 5 μM U0126 or DMSO and inhibitor removed by filtration. Transwell migration assays were performed with this media and MCF10A cells (Left; U, unfiltered medium; F, filtered medium). To show inhibition of Erk phosphorylation by U0126 in the cells used to make the media, they were lysed after media collection and pErk1/2 levels were analyzed by immunoblot (Center). To show that migration inhibition was not due to carryover of U0126 after filtration, MCF10A cells were pretreated for 15 min with conditioned medium or fresh U0126 and were stimulated for 15 min with 20 ng/ml EGF. Lysates of these cells were analyzed by immunoblotting pErk1/2 (Right; U, unfiltered medium; F, filtered medium).