Fig. 9

Inhibition of Src family kinases prevents Dox-induced invasion/migration. MCF7 cells were treated with vehicle (DMSO), Dox (0.4 μM), and Dasatinib as shown. A Protein was extracted and immunoblotted for phospho-Src (Y416), total Src and actin. B, C Cells were seeded in 24-well plate, and a scratch was introduced in each well prior to treatment. B Random field-of-view of wound healing for each condition shown. C Quantification of area of wound (Mean ± SEM, **p < 0.005 vs. veh, n = 3 in triplicate). D Cells in 6-well trays were treated as shown and viable cell number assessed by MTT assay at the time points indicated (Mean ± SEM, **p < 0.01 ***p < 0.001 vs. veh, n = 3). E Cells were treated for 24 h as shown prior to trypsinization and re-seeding into Boyden chambers with 0.8-μm pores with Matrigel overlay. 10% FBS media was used as chemoattractant. Migrated cells were stained with calcein AM dye and detected by fluorescence. Data shown as mean relative fluorescent unit (Mean ± SEM, ***p < 0.001 vs. veh/Dasatinib, n = 3 in triplicate). F, G MCF7 cells were treated with siRNA, vehicle (DMSO), and 0.4 μM Dox as shown. Cells treated with AS negative control, Fyn, or Yes siRNA, were trypsinized and re-seeded in serum-free media into Boyden chambers with 0.8-μm pores, with 10% FBS media as chemoattractant. Migrated cells were stained with crystal violet and random brightfield images were taken with EVOS microscope. Migrated cells were quantified from 5 random fields-of-view for F Fyn and G Yes (Mean ± SEM, **p < 0.01 vs. si-AS, n = 3 in duplicates). All treatments are at 0.4 μM Dox and 0.1 μM Dasatinib unless otherwise noted